Antibiotic-loaded polymeric bone cement is used in orthopedic surgery to deliver local high concentrations of antibiotics to the tissues. The precise mechanism by which antibiotic is released from the polymeric matrix is still not very well known. This research was conducted to investigate the in vitro drug release behavior of antibiotic from acrylic bone cement. A spectrophotometric method for the quantitative analysis of gentamicin sulfate using o-phtaldialdehyde as a derivatizing reagent was thoroughly validated, in order to assure a minimum quality level of the measures. The method proved to be quick, reliable, less expensive than methods such as polarization fluorescence immunoassay and others, and therefore more convenient for the routine analysis of the numerous samples generated during in vitro liberation assays. The release of gentamicin from commercial CMW1® acrylic bone cement samples was investigated following proposed in vitro release experiments.