AbstractA method using ninhydrin reagent for proteolytic activity measurement and kinetic studies with protein or modified protein as substrate was explored. As the reagent is specific for amino group, quantitative expression can be made with the number of peptide bonds cleaved. The sensitivity of the method made it possible for the determination of various kinetic parameters such as Km, Vmaxand Kcatwith the measurement of initial velocity. As an example, these quantities were determined for pancreas protease with casein andN,N‐dimethylcasein as substrates. This study made clear the possibility of using a common substrate of proteolytic enzymes for kinetic studies and a general reference for activity expression. By introducing the use of initial velocity in the kinetic studies of proteolysis with protein as the substrate, this study also makes clear that the kinetics of the enzymatic proteolysis very well fits the Michaelis‐Menten law instead of the Schütz