Proteolytic enzymes are the most versatile and commercially viable group of enzymes comprising over 65% share in the global enzyme market amongst which alkaline proteases have extensive applications in detergent and leather industry. Current study was designed to assess the potential of an alkaline serine protease from Bacillus subtilis ZMS-2 as a bating agent in leather processing. Initially, the production parameters were investigated through Response Surface Methodology (RSM) using Plackett-Burman Design, which identified substrate, agitation speed and incubation temperature as the most significant factors. The optimal levels of these factors were determined through the Box-Behnken experimental analysis as 0.436% substrate concentration, 36.5 °C incubation temperature and 56 rpm agitation speed. The statistical optimization experiments increased the volumetric production of enzyme by 3.94 times (2246 U mL⁻¹) than the initial titer (571 U mL⁻¹). The enzyme was partially purified and characterized as metal ions and detergent compatible serine protease having optimum activity at pH 8 and 60 °C. During the pilot-scale application as a bating agent, the enzyme (340 U mL⁻¹) successfully removed the hair roots and other unwanted proteins from goat skins as observed during scudding and confirmed through Scanning Electron Microscopy. The processed skins displayed enhanced porosity, thumb impression, smoothness and pliability. These findings provide a strong basis for the use of this protease as an efficient and eco-friendly alternative for bating of animal skins in leather tanneries.