This study was carried out to apply an optimized convenient assay, exploiting azo dye-bound chromogenic substrates, to measurement of protease activity. When determined for responses at varying concentrations of two substrates, azocasein and azoalbumin, using 0.5 and 5.0 mg/mL each of bovine pancreas trypsin, 3% azocasein was found to be the most appropriate substrate solution to measure protease activity. Compared with a conventional casein-Folin phenol assay, the chromogen-based protease assay exploiting 3% azocasein showed better precision to have a coefficient of variability in seven repetitive measurements less than 1.11%. When various reagent-grade and industrial proteases that showed proteinase or peptidase activities were tested by this assay at increasing enzyme concentrations, typical shape of rectangular hyperbola in activity-enzyme concentration profiles was observed. In addition, the assay of this study was suitable for activity measurement in real samples that were prepared by hydrolyzing wheat gluten and anchovy fine powder with proteases.