Extract of Withania coagulans fruit was evaluated for milk clotting activity. The highest enzyme activities were detected at pH 4 and 65°C. A 50 mM CaCl₂ was considered sufficient to decrease the milk clotting activity to an effective level. Rennet coagulation time was steadily increased with increasing NaCl and decreasing enzyme concentrations. The enzymatic activity of extract was completely inhibited by pepstatin-A, an aspartic protease inhibitor. Proteomic and zymographic analysis discovered an aspartic protease with Mw of about 35 KDa and pI of 5.91. Our data demonstrated that use of this plant in traditional cheese making has the scientific bases due to existence of an effective protease.