In orthopaedic applications, allografts are used for restoration of bone defects. In order to combine the effects of bone repair and to prevent the infection, antibiotic-impregnated bone grafts are under current investigation with promising early results. In this study, to preserve the stability of antibiotics and to provide appropriate release profiles for 4 - 6 weeks, antibiotic-loaded microspheres were administered in combination with allografts and vancomycin was the antibiotic loaded to microspheres. Particle size, surface characteristics, loading capacity and in vitro release characteristics of the microspheres with and without allografts were determined. In vivo studies were performed on rabbits and antibiotic amount was determined by a fluorescence polarization immunoassay (FPIA) method from synovial fluid sample aspirated. According to the results, although the in vitro study demonstrated effective antibiotic release of vancomycin from antibiotic-impregnated allografts for 5 weeks, in vivo conditions led to an early instability of the antibiotic (in powder form) and contrary to the high initial loading dose an effective release could not be obtained from the allografts after the first week. Following these studies, it was determined that antibiotic release over a minimum inhibitory concentration (MIC) for 6 weeks was realized from vancomycin-loaded microspheres which were implanted in a blend with allografts in bone defects. In conclusion, preservation of the antibiotic in microspheres maintained the bioactivity and provided the controlled antibiotic release, thus implantation of microspheres in a blend with allografts seemed to be a promising carrier system for the orthopaedic applications.