Proteolytic enzymes are one of the significant commercially manufactured enzymes. The manufacture of extracellular alkaline protease by MTCC5152 was explored using several agricultural by-products as substrates viz., cottonseed meal, wheat bran, skimmed milk and soya flour in submerged fermentation, were found to be efficient for enzyme production and commercially significant. Response surface methodology (RSM) is a statistics-based experimental design, sourced to explore the impact of physical parameters on the manufacture of protease from in a batch stirred tank bioreactor (STBR). The four substantial variables (pH, temperature, inoculum size, and agitation) were carefully chosen for optimization analyses and the statistical pattern was created using a central composite design and the quadratic model has been developed. The optimum conditions for protease production (1.51 U mL) where: pH 6.4, temperature 27 °C, inoculum size 2.6%, and agitation 327 rpm. The analysis revealed that the anticipated values were in accord with trial data with a correlation coefficient of 0.969.