Prostaglandin E2 (PGE2) is involved in angiogenesis, bone repair and cartilage metabolism. Thus, PGE2 might represent a suitable signaling molecule in different tissue engineering applications. PGE2 also has a short half-life time. Its incorporation into poly-D,L-lactide-co-glycolide (PLGA) microspheres was demonstrated in a previous study. However, the stability of bioactive PGE2 in these microspheres is unknown. With an adjusted mass spectrometry assay we investigated the amount of incorporated PGE2 and the stability of PGE2 in conventional cell culture medium and in PLGA microspheres. The stability of PGE2 was closely pH dependent. Strong acidic or basic environments reduced the half-life from 300 h (pH 2.6–4.0) to below 50 h at pH 2.0 or pH 8.8. The half-life of PGE2 incorporated into poly-D,L-lactide-co-glycolide increased drastically to 70 days at 37°C and to 300 days at 8°C. Analysis with scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrated a distinct nanostructure of the polymeric phase and both nano- and microporosity.