Food processing technology such as protein hydrolysis using proteases has been receiving a lot of attention, and it is important to accurately understand the cleavage specificity of each protease for selecting a protease suited to aims. Although numerous methods have been reported to reveal the substrate specificity of proteases, there is no method to evaluate simply, quickly, reasonably, and accurately. This study set out to devise Pep-MS assay, a novel assay system that can be used to comprehensively clarify positions at which proteases cleave, by combining a mass spectrometer and a photo-cleavable peptide array. First, we evaluated peptide array corresponding to the primary sequences of alpha(S1)-casein, alpha(S2)-casein and beta-casein with trypsin to verify the accuracy of the Pep-MS assay. The evaluation of cleavage positions by the trypsin protease reagent using the Pep-MS assay resulted in a matching rate of about 96.8% to rational cleavage positions. Next, we confirmed the cleavage positions in alpha(S2)-casein or beta-lactoglobulin by an industrial bacterial protease from Bacillus subtilis at some protease reaction temperatures or reaction times. The Pep-MS assay clarified the differences in the cleavage patterns due to the reaction temperature, and the change in the cleavage strength with the reaction time. Pep-MS assay is a promising method for evaluating the substrate specificity of proteases, which will be useful to find effective production conditions for functional peptide from foods and effective hydrolysis conditions for decreasing allergen of food proteins. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.