This study was intended to formulate, characterize, evaluate in-vitro formulation release and dissolution studies of hydroxyapatite included microspheres of an antimicrobial drug for the disease osteomyelitis. Levofloxacin stacked hydroxyapatite microspheres were set up by inotropic gelation strategy by varing the measure of polymer and carrier. The microspheres of levofloxacin which was formulated were exposed to different physico-chemical parameters like size distribution, solubility studies, FT-IR, drug content, in-vitro dissolution studies, DSC and crystal morphology by SEM. FT-IR results has identified no traces of chemical interaction between the polymer and drug. SEM studies determined that the microspheres were framed in spherical and round shape. DSC curve demonstrated that the melting point of the polymer isn't influencing the melting point of the drug and affirmed that no chemical change among drug and polymer. Anti-microbial activity of formulated microspheres shown a substantial zone of inhibition and inhibition of bacterial growth observed. The in-vitro levofloxacin release from optimized microspheres was initially slow and sustained over the period of time and the cumulative release was 97% in SF-2 and 91% in CF-3 formulation. The drug release pattern was co-related well with Korsmeyer-Peppas model and was followed by case II non fickian release mechanism.