The son of biologists, Northrop was born in Yonkers, New York, and educated at Columbia, obtaining his PhD there in 1915. In 1917 he joined the Rockefeller Institute of Medical Research, only leaving on his retirement in 1961.

In the early 1930s Northrop confirmed some earlier results of James Sumner. Between 1930 and 1935 he and his coworkers succeeded in isolating a number of enzymes, including pepsin, trypsin, chymotrypsin, ribonuclease, and deoxyribonuclease, crystallizing them and exhibiting unequivocally their protein nature. This was sufficient finally to convince chemists that Sumner was correct, and Richard Willstätter had been wrong in his assertion that enzymes are nonprotein.

Using Northrop's techniques, Wendell Stanley was able in 1936 to isolate and crystallize the tobacco mosaic virus, and showed it to be composed of nucleoprotein. Subsequently (1938) Northrop isolated a bacteriophage (bacterial virus) and demonstrated that this also consisted of nucleoprotein. For such work on the isolation and crystallization of proteins and viruses, Northrop, Sumner, and Stanley shared the 1946 Nobel Prize for chemistry.