Leavitt began work in 1893 at Harvard College Observatory as one of the women human ‘computers’ brought in by Edward Charles Pickering to measure and catalog the brightness of stars in the observatory's photographic plate collection. (In the early 1900s, women were not allowed to operate telescopes). She noted thousands of variable stars in images of the Magellanic Clouds. In 1908 she published her results in the Annals of the Astronomical Observatory of Harvard College, noting that a few of the variables showed a pattern: brighter ones appeared to have longer periods. After further study, she confirmed in 1912 that the variable stars of greater intrinsic luminosity – actually Cepheid variables – did indeed have longer periods, and the relationship was quite close and predictable.

Leavitt's discovery is known as the 'period - luminosity relationship' which is a curvilinear relationship that becomes a straight line relation between the luminosity and the log of the period. "A straight line can be readily drawn among each of the two series of points corresponding to maxima and minima," Leavitt wrote of her study of 1,777 variable stars recorded on Harvard's photographic plates, "thus showing that there is a simple relation between the brightness of the variable and their periods".

Leavitt used the simplifying assumption that all of the Cepheids in the respective Magellenic Clouds were approximately the same distances from the earth, so the relationship became key in determining Cepheid Scale Distance and absolute magnitudes beyond the realm of parallax measurement, once the absolute distance to one Cepheid could later be determined by astronomers. "Since the variables are probably at nearly the same distance from the Earth, their periods are apparently associated with their actual emission of light, as determined by their mass, density, and surface brightness." This relationship would provide an important yardstick for measuring distances in the Universe, if it could be calibrated. One year after Leavitt reported her results, Ejnar Hertzsprung determined the distance of several Cepheids in the Milky Way, and with this calibration the distance to any Cepheid could be determined.

At the time, it was not clear that there were millions of nebulae that were actually galaxies outside of our Milky Way galaxy. Their distances were too extreme to be measured using parallax and the Cepheid period - luminosity relationship provided the key to estimating these distances. Cepheids were soon detected in other galaxies such as the Andromeda Galaxy (notably by Edwin Hubble in 1923 – 24). Cepheids were an important part of the evidence that galaxies are far outside of the Milky Way and were key to settling the Great Debate as to the nature of spiral nebulae and whether the Universe was larger than the Milky Way. Our picture of the universe was changed forever, largely because of Leavitt's discovery.

The accomplishments of Edwin Hubble, renowned American astronomer, were made possible by Leavitt's groundbreaking research and Leavitt's Law. "If Henrietta Leavitt had provided the key to determine the size of the cosmos, then it was Edwin Powell Hubble who inserted it in the lock and provided the observations that allowed it to be turned," wrote David H. and Matthew D.H. Clark in their book Measuring the Cosmos. To his credit, Hubble himself often said that Leavitt deserved the Nobel for her work. Gösta Mittag - Leffler of the Swedish Academy of Sciences had begun paperwork on her nomination in 1924, only to learn that she had died of cancer three years earlier (the Nobel prize cannot be awarded posthumously).

Leavitt worked sporadically during her time at Harvard, often sidelined by health problems and family obligations. An illness contracted after her graduation from Radcliffe College rendered her increasingly deaf. By 1921, when Harlow Shapley took over as director of the observatory, Leavitt was made head of stellar photometry. By the end of that year she had succumbed to cancer, and was buried in the Leavitt family plot at Cambridge Cemetery in Cambridge, Massachusetts.

"Sitting at the top of a gentle hill," writes George Johnson in his biography of Leavitt, "the spot is marked by a tall hexagonal monument, on top of which (cradled on a draped marble pedestal) sits a globe. Her uncle Erasmus Darwin Leavitt and his family are also buried there, along with other Leavitts. A plaque memorializing Henrietta and her two siblings who died so young, Mira and Roswell, is mounted directly below the continent of Australia. Off to one side, and more often visited, are the graves of Henry and William James."

Leavitt was a member of Phi Beta Kappa, the American Association of University Women, the American Astronomical and Astrophysical Society, the American Association for the Advancement of Science, and an honorary member of the American Association of Variable Star Observers. Her early passing was seen as a tragedy by her colleagues for reasons that went beyond her scientific achievements.

In an obituary her colleague, Solon I. Bailey, noted, "She had the happy faculty of appreciating all that was worthy and lovable in others, and was possessed of a nature so full of sunshine that, to her, all of life became beautiful and full of meaning."

• The asteroid 5383 Leavitt and the crater Leavitt on the Moon are named in her honor.
• Unaware of her death four years prior, the Swedish mathematician Gösta Mittag - Leffler considered nominating her for the 1926 Nobel Prize in Physics, and wrote to Shapley requesting more information on her work on Cepheid variables, offering to send her his monograph on Sofia Kovalevskaya. Shapley replied, let Mittag - Leffler know that Leavitt had died, and suggested that the true credit belonged to his (Shapley's) interpretation of her findings. She was never nominated, because the Nobel Prize is not awarded posthumously.