Gelatinase B is a member of the metalloproteinase family of enzymes that degrade the extracellular matrix under normal and pathological conditions, including autoimmune diseases and tumor cell dissemination. In the present work, we describe a simple and reliable method that allows qualitative and quantitative measurements of a specific enzymatic reaction (mediated here by gelatinase B) by flow cytometry using fluorochrome-labeled substrate coated on polystyrene microspheres. Using this approach, proteolytic degradation can be monitored by the decrease of the fluorescence emitted by the microspheres following incubation with the enzyme. In most of the experiments, the signal-to-noise ratio between autofluorescent microspheres and those coated with the fluorescein isothiocyanate (FITC)-labeled substrate was near 500. This ratio allows one to measure accurately the enzymatic activity in the presence of chemical or biological inhibitors. FITC labeling and passive adsorption of substrates on the solid support did not cause significant conformational changes or steric hindrance that would interfere with the proteolytic activity of gelatinase B. This method constitutes a powerful tool for the measurement, on a routine basis, of the net activity resulting from the balance between the gelatinase B activity and the presence of natural inhibitors and for the identification of new metalloproteinase inhibitors that could suppress the excessive proteolytic activity that characterizes many disease processes.