Factors affecting milk clotting activity of rennet substitute extracted from green bracts of artichoke (Cynara scolymus) flowers in different buffer solutions were evaluated. These factors include extraction pH, clotting temperature, extract concentrations, addition of CaCl₂, NaCl and glucono delta-lactone (GDL) at different concentrations. The proteolytic activity of different extract solutions and rheological properties of produced curd was studied as well. The obtained results indicated that the optimum clotting activity of artichoke crude extracts improved water holding capacity and susceptibility to syneresis obtained at pH value of 5.0-6.0, temperature of 65-70°C and 3%, 0.04-0.05, 3%, 0.5% of crude extract, CaCl₂, NaCl and GDL concentrations, respectively. The optimum clotting activity of artichoke crude extracts indicated that the sodium acetate buffer solution T4 (5% NaCl in sodium acetate buffer, pH 5.0) and sodium phosphate buffer, T5 (5% NaCl in sodium phosphate buffer, pH 5.5), T6 (5% NaCl in sodium phosphate buffer, pH 6.5), and T7 (5% NaCl in sodium phosphate buffer, pH 7). Results also indicated that artichoke crude extracted in sodium phosphate buffer solutions and sodium acetate buffer (T4) had higher proteolytic activity than other buffers. The best rheological properties of resultant curds were noticed in sodium phosphate buffers (T5, T6, T7) and sodium acetate buffer (T4).