Proteases are unique class of industrial biocatalyst; occupy a key chair with respect to wide range of utility in both physiological and commercial sector. Repertoire of various enzyme assays were suggested for determining enzyme units of protease. Majority of currently reported assays are expensive, time consuming and cumbersome. In this direction, a development of speedy and sensitive quantitative enzyme assay for the characterization of proteases is highly desirable. The present investigation reported a quick method for characterization of proteases using single substrate-agarose plate method. A trypsin (obtained from bovine pancreas) and bacterial protease (serine protease of Bacillus circulans MTCC 7942) on subsequent interaction with proteinaceous substrate gives proteolytic zone on substrate-agarose plate. The clear zone diameter of proteolysis was directly proportional to enzyme units of protease. Furthermore, enzyme activities of protease treated with various chemical activator/inhibitor gives vis-à-vis response and yielded significant data. The biochemical characteristics of proteases are documented using rapid single substrate-agarose plate method. A rapid, sensitive and reliable improved quantitative protease assay using single substrate-agarose plate method could be used at academic, research and commercial level.