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Introduction:In the recent years, a growing interest for the production of secretary recombinant proteins is seen. This is due to the advantages of recombinant protein production in the periplasm compared to the cytoplasm. However, signal peptides have a critical role in protein secretion as well as the applied technique for the extraction of the protein. Granulocyte colony stimulating factor (GCSF) is a type of colony stimulating factor that causes motivating of proliferation, differentiation and survival of neutrophiles and progenitor cells of these cells and are used to promote decreased neutrophils in some cancers after chemotherapy. The aim of this study was to evaluate the usage of different osmotic shock assays in order to achieve the highest amount of granulocyte colony stimulating factor (GCSF) in BL21 strain of E.coli. Materials and methods: The E.coli which contained pET22b- GCSF2- Intein2 expression vector was cultured in 4YT medium and was induced with IPTG 1Mm for protein production. It is necessary to mention that the pET22b has a pelB signal peptide that directs proteins to the periplasmic space. In the next step, three different methods of osmotic shocks were applied for the extraction of the obtained human recombinant protein. Finally, the isolated proteins were analyzed by SDS-PAGE and western blot techniques. Results: The results of this investigation indicated that GCSF was produced in both of the cytoplasmic and periplasmic spaces and the best method of osmotic shock for protein extraction is using Tris buffer and MgSO4. Discussion and conclusion: Regarding the results, it is concluded that the MgSO4 with Tris buffer create a good osmotic pressure and accordingly is a more effective way for G-CSF protein extraction. As a result, this method could be used for production and simple separation of recombinant drug proteins.