Atomic force microscopy (AFM) is recognized a suitable and powerful technique for surface and morphological analysis. Even if until now this technique has not been frequently used in the pharmaceutical field, it can contribute to an accurate morphologic characterization of microspheres and nanospheres. In this work, atomic force microscopy has been used to perform the surface characterization of sterilized microspheres. The aim is to investigate the morphologic modifications induced by gamma irradiation on poly(lactide-co-glycolide) microspheres loaded with ovalbumin and to compare the results obtained by AFM to those obtained by scanning electron microscopy (SEM). The results obtained show that, with respect to SEM, AFM can give some additional information regarding the modifications induced by gamma-irradiation on microspheres surface morphology. The significant changes in surface roughness after irradiation are indicative of damage due to gamma-irradiation. The unchanged surface roughness values calculated for microspheres containing PEG in their matrix, suggest that this polymer exerts a protective effect towards gamma-irradiation.