Prolidase deficiency (PD) is a rare autosomal recessive disorder caused by inadequate levels of the cytosolic exopeptidase prolidase (E.C. 3.4.13.9), for which there is not, as yet, a resolutive cure. We have investigated whether biodegradable microspheres loaded with prolidase could release active enzyme inside cells, to consider this system as a possible therapeutic approach for prolidase deficiency. Poly(lactide-co-glycolide) microspheres were prepared, modifying the classical double emulsion solvent evaporation method to mitigate the burst effect of the enzyme from the microspheres. Ex-vivo experiments were performed, by incubating microencapsulated prolidase with cultured fibroblasts from PD patients and from controls, to determine the amount of active enzyme delivered to the cells. The microparticulate drug delivery system described carried small amounts of active prolidase inside fibroblasts, ensuring a response to the intracellular accumulation of X-Pro dipeptides, the mechanism that is supposed to be responsible for the development of clinical manifestations of this disorder in man. A positive result of the presence of active enzyme inside cells was an improvement in fibroblast shape.