Prout was born at Horton in England and studied medicine at Edinburgh, graduating in 1811. He established himself as a physician in London and became a pioneer of physiological chemistry, in which he lectured. He wrote on the stomach and urinary diseases and on the chemistry of the blood, urine, and kidney stones. In 1818 he prepared urea for the first time and in 1824 he identified hydrochloric acid in stomach secretions. He was also one of the first to divide food components into the groups of fats, carbohydrates, and protein.

Prout's fame also rests on a paper he published anonymously in 1815, On the Relation between the Specific Gravities of Bodies in Their Gaseous State and the Weight of Their Atoms. In this he formulated what has since been called Prout's hypothesis: the atomic weight of all atoms is an exact multiple of the atomic weight of hydrogen. Determination of atomic weights had made this view plausible. At the time there was considerable interest in the hypothesis as it implied that elements were themselves ‘compounds’ of hydrogen, and Prout suggested that hydrogen was the prima materia (basic substance) of the ancients. However, more accurate determinations of atomic weight, particularly by Jean Stas, showed that many were not whole numbers. Stas described the hypothesis as “only an illusion” although he also remarked that there was “something at the bottom of it.” Interest was revived with the publication of Dmitri Mendeleev's periodic table, although Mendeleev described the idea of a prima materia as “a torment of classical thought.” The discovery of isotopes in the 20th century resolved the position.