Matrix metalloproteinases (MMPs) are a family of enzymes that are up-regulated in many diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). Here we report on a novel technique that can be used to simultaneously measure activity levels for a panel of enzymes, such as the MMPs. The technique, termed the multiple-enzyme/multiple-reagent assay system (MEMRAS), relies on the use of reagents such as substrates with varying selectivity profiles against a group of enzymes. When reaction rates are measured by following a change in fluorescence with time, for mixtures of enzymes, an equation with unknown concentrations for each activity is generated for each reagent used. Simultaneously solving the set of equations leads to a solution for the unknown concentrations. We have applied this mathematical technique to measure activity levels for mixtures of MMPs such as collagenase 3 and gelatinase A. In addition, because we were most interested in determining collagenase 3 levels as a potential biological marker for OA, we developed highly selective substrates for this enzyme by using results found in previous bacteriophage substrate-mapping experiments. Some of the best substrates tested have specific activities for collagenase 3 that are 37,000-, 17,000-, 90-, and 200-fold selective over stromelysin 1, collagenase 1, and gelatinases A and B, respectively.