Bovine serum albumin, lysozyme, and trypsin inhibitor were first encapsulated into pory-d,l-lactide-co-glycolide (PLGA) microspheres and then a new strategy was used to quantitate the actual levels of proteins in the microspheres. The proper combination of water-miscible dimethyl sulfoxide and 0.05 N-NaOH containing 0.5% sodium dodecyl sulfate (SDS) made it possible to solubilize both PLGA microspheres and proteins in a single phase. A total protein assay conveniently provided accurate information on the amount of protein encapsulated into the microspheres. In contrast to conventional techniques making use of acetonitrile, dichloromethane and SDS extraction methods, this new method did not necessitate polymer precipitation, filtration, and protein extraction into other phases. These features were a great advantage in recovering proteins without any loss due to experimental processes. As a consequence, the new method reported in this study provided accurate data for the actual level of protein in PLGA microspheres, regardless of the pattern of protein distribution inside microspheres or the characteristics of microspheres. The experiment relying on the use of a radiolabeled protein also validated the reliability of this new method.