Cellular homeostasis is maintained by a complex network of reactions catalyzed by enormous numbers of enzymatic activities (the enzymome), which serve to determine the phenotypes of cells. Here, we focused on the enzymomics of proteases and peptidases because these enzymes are an important class of disease-related proteins. We describe a system that (A) simultaneously evaluates metabolic activities of peptides using a series of exogenous peptide substrates and (B) identifies the enzymes that metabolize the specified peptide substrate with high throughput. We confirmed that the developed system was able to discover cell-type-specific and disease-related exo-and endopeptidase activities and identify the responsible enzymes. For example, we found that the activity of the endopeptidase neurolysin is highly elevated in human colorectal tumor tissue samples. This simple but powerful enzymomics platform should be widely applicable to uncover cell-type-specific reactions and altered enzymatic functions with potential value as biomarkers or drug targets in various disease states and to investigate the mechanisms of the underlying pathologies.