The method of determining the enzymatic activity of acid proteinases is described. The method is based on the use of /sup 125/I-labelled natural protein substrates. /sup 125/I-labelled albumin, /sup 125/I-globulin and /sup 125/I-insulin were tested for the determination of activities. All the substrates were hydrolyzed with the enzymes of the supernatant fraction (106,000 g) of beef liver homogenate in the acid pH zone. Optimum enzymatic reaction conditions were tested, the dependence of the reaction on the enzyme concentration, time and temperature was determined, pH optimum was ascertained for the individual substrates, and pH stability was determined. The results show that the method is suitable for determining the enzymatic activity of proteinases of cathepsin character.