Microbial proteases are more economical than plant- and animal-derived proteases due to their ease of production and high activity. This study aimed to optimize the production of proteases from fermentative food-derived microorganisms. Five strains with proteolytic activity among 50 Bacillus sp. were first screened. Two strains with high protease activity were identified: Bacillus amyloliquefaciens SRCM 102139 and Bacillus subtilis SRCM 104999. SRCM 102139 strain and SRCM 104999 strain had the highest protease activity in 0.8% glucose and 0.3% yeast extract, and in 0.8% starch and 0.1% soy peptone, respectively. The production of protease for two strains was optimized by the Central Composite Design (CCD) under response surface methodology. The optimal conditions for protease production in SRCM 102139 were 0.5% and 0.347%, pH 6.0, for carbon (glucose) and nitrogen (yeast extract springer 0202) sources, respectively, with a predicted value of 0.929 U/mL. Additionally, the optimal conditions for protease production in SRCM 104999 were 0.5% and 0.5%, pH 6.7, for carbon (starch) and nitrogen (soy peptone HSP-349) sources, respectively, with a predicted value of 0.431 U/mL. The actual protease activities of SRCM 102139 and SRCM 104999 under the established conditions were 0.926 U/mL and 0.428 U/mL, respectively, closely matching the predicted values.