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Optimization of the growth condition for maximum growth rate and protease production was carried out using Bacillus subtilis. The optimization of protease production using agro-industrial waste product such as cassava waste as substrate was performed with statistical methodology based on experimental designs. The screening of twelve nutrients for their influence on protease production was achieved using a Plackett-Burman design. MgSO4•7H2O, casein and glucose were selected based on their positive influence on protease production. The selected components were optimized using response surface methodology (RSM). The optimum conditions are (% w/w): MgsO4•7H2O - 0.14, casein - 1.4 and glucose - 2.64. These conditions were validated experimentally which revealed an enhanced protease yield of 202.048 U/gds.