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Gentamicin sulfate (GS)-loaded poly(L-lactide-co-glycolide) (PLGA) microspheres (MSs) were prepared by O/O solvent evaporation method for the sustained local antibiotic delivery. We have investigated the effects of initial drug loading, emulsifier amount, polymer concentration, and amount of continuous phase on the release profile. The morphology of GS/PLGA MSs was examined with scanning electron microscope (SEM) before and after in vitro release test for the explanation of biodegradation mechanism of GS/PLGA MSs. The loading efficiency and in vitro release of GS were analyzed by high performance liquid chromatography (HPLC) and the released GS activity was determined by microbiological assay using Staphylococcus aureus. The increasing polymer concentration and emulsifier amount lead to the increase in loading efficiency. The release profiles from the GS/PLGA MSs could be controlled by varying preparation conditions such as initial drug loading, emulsifier amount, polymer concentration, and amount of continuous phase. We investigated the antibiotics impregnated biodegradable devices to reach high local drug levels while maintaining low systemic levels of antibiotics, with the near-zero order release patterns of drug for 2, 3, 10 and 45 days from PLGA MSs.