To maximize the efficiency of utilization of a pancreatic enzyme cocktail and estimate the contamination for in vitro protein digestibility assays, the specific activity losses of trypsin and chymotrypsin and the digestion of the enzyme proteins were studied. In the absence of protein substrate, increase of enzyme concentration augmented the half‐lives of trypsin and chymotrypsin and decreased the digestion of enzymatic proteins. In contrast, in the presence of substrate, increase of enzyme concentration decreased trypsin's half‐life. Increase of pH augmented the digestion of enzymatic proteins. The results indicated the optimum time for utilization of the enzymes depended on pH, enzyme concentration and presence of substrate. At the time when digestion of the proteins ceased, the average size of the hydrolysates was calculated between 3.1 and 5.4 amino acid residues, suggesting most proteins in the enzyme cocktail would be detected as digestible proteins.