Abstract: The aim of present study was to apply experimental design methodology in the development and optimization of carrier system for Serratiopeptidase by modified double emulsion solvent evaporation technique. A three-level three-factorial Box-Behnken experimental design was used to characterize and optimize three physicochemical parameters-Eudragit RS100 concentration, external aqueous phase volume and stirring speed of primary emulsion on the entrapment and size of microspheres. The Response Surface Methodology (RSM) and multiple response optimization utilizing the polynomial equation were used to select optimal formulation with maximum entrapment and particle size in range. The maximum entrapment (80.62±1.96%) was achieved with 300 mg Eudragit RS100, 100 mL EAP and 2000 rpm as stirring speed and the observed responses coincided well with the predicted values from the RSM optimization technique. In vitro proteolytic activity confirmed the bioactivity of peptide after microencapsulation. The drug release from formulations showed a similar sustained release showing an initial burst followed by diffusion. In conclusion, a novel, controlled-release delivery system for peptide drug was successfully developed by experimental design methodology with the fewest number of experiments.