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Protein A is found in the cell wall of bacterium Staphylococcus aureus. It can be used in the purification process of monoclonal and polyclonal antibodies. Enzymatic method using Lysostaphin Enzyme is the most common and standard method for the isolation of protein A. Never the less this method is very expensive in an industrial Scale. Hereby, we have developed an alternative method to isolate this protein without Lysostaphin digestion, in a time and cost effective manner. Five ways Hot Acid, Freeze and Thaw, Toluene- Triton×100-EDTA, Lysozyme and Sodium Meta Priodate were used to lysis of cell wall bacteria were performed. Here in this study we compared five mentioned method to achieve bacterial cell wall lysis. The obtained results were compared to the standard Lysostaphin digestion method. In all of the studies Staphylococcus aureus strain NCTC 8325 was used for protein A extraction. Bacterial cell wall 53 KD a protein A, extracted from the mentioned strain, was purified by ammonium sulfate (80%) precipitation method. The results were analyzed and confirmed via Gram staining for microscopic evaluations, SDS-PAGE Analysis, Validated Lowry assay, ELISA Test with both human and goat serum samples, and western blotting with rabbit IgG. The obtained data were compared to assess the yield of protein extraction. The results revealed that 300µg protein A per 300g wet weigh of Cowan strain was extracted via lysozyme digestion method, compared to the developed method, 283.7 µg protein A per 50g wet weigh of the same strain. This amount approximately 6 fold increased which is statistically significant. According to the obtained results stated above, we can conclude that utilization of lysozyme digestion method following ammonium sulfate 80%, Precipitation method is and effective method in the terms of cost and ease of industrial production line design.