Abstract The Bratton-Marshall test for naphthylamine has been modified and adapted to determine the naphthylamine released on hydrolysis of the specific chymotrypsin substrate glutaryl- l -phenylalanine-β-naphthylamide. This substrate shows advantages over others used in similar procedures because it combines satisfactorily solubility, specificity, and sensitivity with the release of naphthylamine upon hydrolysis. The procedure has been applied to the determination of chymotrypsin itself and to other enzymes with similar specificities such as the chymotrypsinlike cathepsin C of liver, kidney, tumors, and other tissues. It has been used also to assay chymotrypsin inhibitors, particularly those found in tissues.