Objective: The main aim of the study was to screen, optimize and partial purification of protease from fungi. The isolated fungi were screened for protease activity on a modified SDA plate and subjected to media optimization. Comparative study of free and immobilized cells was done. Methods: A partial sequencing of the fungi was done. The process parameters were examined by the classical method and Plackett Burman method, including carbon, nitrogen sources, pH, temperature, agitation and inoculum size were optimized. The enzyme was subjected to step by step purification process by ammonium sulfate precipitation. Results: The total protein concentration was determined and the specific activity was also performed by casein hydrolysis assay. The Plackett-Burman method shows fructose, peptone pH are significant factors for optimized protease production. Immobilized cell shows reduction in protease production. In optimum conditions the protease produced was 4758.4U/mg. Protease was partially purified at 60% ammonium sulfate saturation. Conclusion: Trichosporon japonicum VITVK1 estimated as a strong candidate for the production of Protease. © 2015, International Journal of Pharmacy and Pharmaceutical Sciences. All rights reserved.