The β- and γ-irradiation effects on stability of microspheres made of poly(lactide-co-glycolide) 50:50 copolymer (PLGA) containing bupivacaine (BU) were studied. Microspheres containing 10, 25, and 40% w/w, respectively, of BU were prepared by spray drying and irradiated in air with β- and γ-irradiation at a dose of 25 kGy. Morphology (atomic force microscopy, particle-size analysis), physico–chemical characteristics (DSC and FT-IR spectroscopy), drug content and in vitro dissolution profile of microspheres were all determined; the stability of irradiated microspheres was evaluated over a 9-month period. The decrease of BU content in γ-irradiated microspheres was almost always constant independent of the amount of BU per sample, therefore it was in inverse proportion to drug loading (range between 5 and 15%). BU release rate increased immediately after irradiation and increased slightly until 90 days of storage. As far as β-irradiated microspheres are concerned, BU content decreased in a significant way (≈3%) only in microspheres containing 10% w/w of BU. Immediately after irradiation, drug release rate in β-irradiated microspheres increased less than in the corresponding γ-irradiated microspheres, and it did not change further over the following storage period. BU-loaded microspheres have been shown to be more stable against β- than γ-irradiation. AFM revealed that the surface roughness of the irradiated microspheres increases depending on irradiation. As such, if a parameter is quantifiable, it is proposed as a marker of degradation due to ionizing radiation.