An extraction and assay system was developed for quantifying endogenous muscle proteases from a single 5-g sample. A single extraction buffer was developed for simultaneous extraction of both calcium-dependent proteases (CDP) and cathepsins. Protease activity determined by the modified procedure was compared to standard procedures currently used in our laboratory. The successful use of the modified procedure on muscle biopsies was verified. Activities per gram of ovine longissimus muscle of CDP system components for 50-g standard and 5-g modified procedures were not different (P greater than .05) for CDP-I (1.16 vs 1.08), CDP-II (.89 vs 1.03), or CDP inhibitor (2.34 vs 2.32), respectively. Activities of cathepsins per gram of muscle for standard and modified procedures were higher (P less than .05) for the modified procedure (cathepsins B + L, 202.0 vs 309.8), but not different (P greater than .05) for cathepsin B (76.6 vs 98.8). Cystatin-like activity was not different (P greater than .05; 3.4 vs 3.2). To test the effect of location within the longissimus muscle on protease activities, 5 g of longissimus muscle was removed immediately postmortem from each of six locations from each side of three steer carcasses. Location within the longissimus muscle had no effect (P greater than .05) on the protease activities measured. Protease activities determined on bovine longissimus muscle biopsies with the modified procedure were similar to immediate postmortem activities. These data verify that the modified procedure was as able to quantify endogenous muscle proteases as the standard procedures and could be used on muscle biopsies. This procedure should be useful in studying the role of endogenous muscle proteases in muscle growth and postmortem proteolysis.