Abstract: The purpose of this study was to develop protein containing microspheres by multiple emulsification (water-in-oil-in-water, w/o/w) technique at different homogenizing speeds using biodegradable polymer poly (D,L-lactide-co-glycolide) (85:15/PLGA). Here, ovalbumin was used as a model protein drug. Although, many studies available with PLGA-based protein loaded microparticles, none of them provides sufficiently convincing technologies for manufacturing protein-loaded microparticles with all the standardized process parameters such as amount of protein loading, protein-release, stability of the protein, polydispersity and zeta potential. Therefore, further research is required in the field. Various process parameters such as protein polymer interaction, particle size, surface morphology, drug loading, protein-polymer ratio, zeta potential, polydispersity, protein stability and protein release characteristics were studied here. In vitro protein release study showed that release profile of ovalbumin from biodegradable microspheres varied due to the change in homogenizing speeds during multiple emulsion preparation technique. Drug loading efficiency varied from 21.82 to 36.54%. The protein biodegradable microspheres were spherical in shape. The stability studies of protein were investigated at different temperatures for 30 days by using a Fourier Transform Infrared (FTIR) spectroscopy following ICH guidelines. The FTIR analysis showed the structural integrity of ovalbumin in PLGA microspheres. PLGA microspheres containing ovalbumin as a model protein could be useful for the controlled delivery of similar protein drugs.