Abstract : The papain-like protease (PLpro) of SARS-CoV2 is an attractive target for the development of novel drugs to combat COVID-19. However, discovering effective drug candidates often requires the production of recombinant PLpro with high purity and yield. Unfortunately, this process often involves automated chromatography systems that require expensive and sophisticated equipment. This study aimed to purify recombinant PLpro using a manual Ni-NTA chromatography system with a step elution approach. Initially, PLpro was expressed in Escherichia coli BL21(DE3) and the soluble fraction was used for purification using a His-Trap column. Elution was performed with an elution solution containing imidazole in the concentration range of 10-500 mM. PLpro was successfully eluted with a minimum of 50 mM imidazole, but the highest purity was obtained at 400-450 mM imidazole. The yield and purification fold of PLpro obtained in this study were approximately 50.54% and 9.30, respectively, with a protein amount of 8.6 mg and a specific activity of 0.55 U/mg. The purified PLpro exhibited normal behavior in terms of its pH and temperature optimum, which were found to be at pH 6-7 and 30°C, respectively. These results indicate that the use of non-automated chromatography and a step elution process is a viable approach for producing recombinant PLpro with acceptable purity.