Boyle returned to England from the Continent in mid 1644 with a keen interest for scientific research. His father had died the previous year and had left him the manor of Stalbridge in Dorset, together with some estates in Ireland. From that time, he devoted his life to scientific research, and soon took a prominent place in the band of inquirers, known as the "Invisible College", who devoted themselves to the cultivation of the "new philosophy". They met frequently in London, often at Gresham College; some of the members also had meetings at Oxford where Boyle went to reside in 1654. Reading in 1657 of Otto von Guericke's air-pump, he set himself with the assistance of Robert Hooke to devise improvements in its construction, and with the result, the "machina Boyleana" or "Pneumatical Engine", finished in 1659, he began a series of experiments on the properties of air. An inscription can be found on the wall of University College, Oxford the High Street at Oxford (now the location of the Shelley Memorial), marking the spot where Cross Hall stood until the early 1800s. It was here that Boyle rented rooms from the wealthy apothecary who owned the Hall.

An account of Boyle's work with the air pump was published in 1660 under the title New Experiments Physico-Mechanicall, Touching the Spring of the Air, and its Effects.... Among the critics of the views put forward in this book was a Jesuit, Franciscus Linus (1595 – 1675), and it was while answering his objections that Boyle made his first mention of the law that the volume of a gas varies inversely to the pressure of the gas, which among English-speaking people is usually called after his name. However, the person that originally formulated the hypothesis was Henry Power in 1661. Boyle included a reference to a paper written by Power, but mistakenly attributed it to Richard Townley. In continental Europe the hypothesis is sometimes attributed to Edme Mariotte, although he did not publish it until 1676 and was likely aware of Boyle's work at the time. In 1663 the Invisible College became the Royal Society of London for the Improvement of Natural Knowledge, and the charter of incorporation granted by Charles II of England, named Boyle a member of the council. In 1680 he was elected president of the society, but declined the honour from a scruple about oaths.

It was during his time at Oxford that Boyle was a Chevalier. The Chevaliers are thought to have been established by royal order a few years before Boyle's time at Oxford. The period of Boyle's residence was marked by the reactionary actions of the victorious parliamentarian forces, consequently this period marked the most secretive period of Chevalier movements and thus little is known about Boyle's involvement beyond his membership.

In 1668 he left Oxford for London where he resided at the house of his sister, Lady Ranelagh, in Pall Mall.

In 1689 his health, never very strong, began to fail seriously and he gradually withdrew from his public engagements, ceasing his communications to the Royal Society, and advertising his desire to be excused from receiving guests, "unless upon occasions very extraordinary", on Tuesday and Friday forenoon, and Wednesday and Saturday afternoon. In the leisure thus gained he wished to "recruit his spirits, range his papers", and prepare some important chemical investigations which he proposed to leave "as a kind of Hermetic legacy to the studious disciples of that art", but of which he did not make known the nature. His health became still worse in 1691, and he died on 30 December that year, just a week after that of the sister with whom he had lived for more than twenty years. Robert Boyle died from paralysis. He was buried in the churchyard of St Martin in the Fields, his funeral sermon being preached by his friend Bishop Gilbert Burnet. In his will, Boyle endowed a series of Lectures which came to be known as the Boyle Lectures.

Boyle's great merit as a scientific investigator is that he carried out the principles which Francis Bacon espoused in the Novum Organum. Yet he would not avow himself a follower of Bacon, or indeed of any other teacher. On several occasions he mentions that in order to keep his judgment as unprepossessed as might be with any of the modern theories of philosophy, until he was "provided of experiments" to help him judge of them, he refrained from any study of the Atomical and the Cartesian systems, and even of the Novum Organum itself, though he admits to "transiently consulting" them about a few particulars. Nothing was more alien to his mental temperament than the spinning of hypotheses. He regarded the acquisition of knowledge as an end in itself, and in consequence he gained a wider outlook on the aims of scientific inquiry than had been enjoyed by his predecessors for many centuries. This, however, did not mean that he paid no attention to the practical application of science nor that he despised knowledge which tended to use.

Boyle was an alchemist; and believing the transmutation of metals to be a possibility, he carried out experiments in the hope of effecting it; and he was instrumental in obtaining the repeal, in 1689, of the statute of Henry IV against multiplying gold and silver. With all the important work he accomplished in physics – the enunciation of Boyle's law, the discovery of the part taken by air in the propagation of sound, and investigations on the expansive force of freezing water, on specific gravities and refractive powers, on crystals, on electricity, on colour, on hydrostatics, etc. – chemistry was his peculiar and favourite study. His first book on the subject was The Sceptical Chymist, published in 1661, in which he criticized the "experiments whereby vulgar Spagyrists are wont to endeavour to evince their Salt, Sulphur and Mercury to be the true Principles of Things.". For him chemistry was the science of the composition of substances, not merely an adjunct to the arts of the alchemist or the physician. He endorsed the view of elements as the undecomposable constituents of material bodies; and made the distinction between mixtures and compounds. He made considerable progress in the technique of detecting their ingredients, a process which he designated by the term "analysis". He further supposed that the elements were ultimately composed of particles of various sorts and sizes, into which, however, they were not to be resolved in any known way. He studied the chemistry of combustion and of respiration, and conducted experiments in physiology, where, however, he was hampered by the "tenderness of his nature" which kept him from anatomical dissections, especially of living animals, though he knew them to be "most instructing".

Besides being a busy natural philosopher, Boyle devoted much time to theology, showing a very decided leaning to the practical side and an indifference to controversial polemics. At the Restoration he was favourably received at court, and in 1665 would have received the provostship of Eton College, if he would have taken orders; but this he refused to do on the ground that his writings on religious subjects would have greater weight coming from a layman than a paid minister of the Church. As a director of the East India Company he spent large sums in promoting the spread of Christianity in the East, contributing liberally to missionary societies, and to the expenses of translating the Bible or portions of it into various languages. He founded the Boyle Lectures, intended to defend the Christian religion against those he considered "notorious infidels, namely atheists, deists, pagans, Jews and Muslims", with the provision that controversies between Christians were not to be mentioned. In 2004, the Boyle Lectures were resurrected in London.