Northrop studied the kinetics of enzyme-catalyzed reactions and the conditions affecting the action of the digestive enzymes pepsin and trypsin. In 1931, he developed solubility methods to determine the homogeneity of protein preparations. These methods helped prove that enzymes are proteins.

Between 1930 and 1935, Northrop crystallized pepsin. His colleague, Moses Kunitz, crystallized trypsin, chymotrypsin, ribonuclease, deoxyribonuclease, hexokinase, and pyrophosphatase and used the most rigorous test then available to demonstrate they, too, are pure proteins.

In later studies, Northrop discovered that bacteria-invading agents known as bacteriophages - long thought of as living organisms - could be isolated as chemical substances. The finding helped prove that bacteriophages are viruses and paved the way for scientists to study viruses in bacteria.

Northrop's findings led him to theorize, correctly, that viruses can transfer genetic information from one cell to another. In 1946, he and Wendell M. Stanley shared half the Nobel Prize "for their preparation of enzyme and virus proteins in a pure form." (James Sumner received the other half of the prize.) The award also recognized the philosophical importance of the link between living and nonliving matter discovered by Stanley.