In biomedical research and clinical diagnostics, it is a major challenge to measure disease-related degradative enzyme activity directly in whole blood, Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time-consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge-changing fluorescent peptide substrates. Charge-changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic alpha-chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both alpha-chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross-reactivity with the trypsin-like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin specific), a detection limit of about 10-20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point-of-care diagnostics.