Using lysozyme as a model protein, this study investigated protein stability, protein–polymer interaction in different release media and their influence on protein release profile and in vitro–in vivo correlation. Lysozyme was microencapsulated into PLGA 50:50 by a double emulsion–solvent extraction/evaporation method. Protein stability, protein–PLGA adsorption and protein in vitro release were studied in various test media. Differential scanning calorimetry analysis showed lysozyme to be most conformationally stable in pH 4.0 acetate buffer with highest T_m at 77.2 °C and ΔH_cal 83.1 kcal/mol. Lysozyme exhibited good stability in pH 2.5 glycine buffer with T_m at 63.8 °C and ΔH_cal 69.9 kcal/mol. In pH 7.4 phosphate-buffered saline (PBS), lysozyme showed a trend toward aggregation when the temperature was elevated. When PLGA polymer was incubated with lysozyme in the various buffers, adsorption was found to occur in PBS only. The adsorption severely limited the amount of lysozyme available for release from microspheres, resulting in slow and incomplete release in PBS. In contrast, the release of the microspheres in acetate and glycine buffers was complete within 40 and 70 days, respectively. Radiolabeled lysozyme blood levels in rats from the microspheres correlated qualitatively well with in vitro release in glycine buffer as a release medium. This study suggests that protein stability and adsorption are critical factors controlling protein release kinetics and in vitro–in vivo correlation of PLGA microspheres.