Proust was born the son of an apothecary at Angers in northwest France. He studied in Paris and became chief apothecary at the Saltpêtrière Hospital. In 1789 he went to Madrid to become director of the Royal Laboratory under the patronage of Charles IV. After the invasion of Spain by Napoleon, the fall of his patron, and the destruction of his laboratory by the invading army, he returned to France in 1808. He lived in poverty for some years before being awarded a pension by Louis XVIII.

In 1799 Proust formulated his law of definite proportions. He pointed out that copper carbonate must always be made from the same fixed proportions of copper, carbon, and oxygen. From this he generalized that all compounds contained elements in certain definite proportions. Proust's law was not immediately accepted by all chemists; in particular, his proposal led to a long and famous controversy with Claude-Louis Berthollet who argued that elements could combine in a whole range of different proportions. It is now clear that Proust was talking about compounds whereas Berthollet was thinking of solutions or mixtures. Berthollet eventually admitted his error.

The strength of Proust's law was seen a few years later when John Dalton published his atomic theory. The law and the theory fitted exactly – Proust's definite proportions being in fact a definite number of atoms joining together to form molecules.