Two methods for the determination of (+)- and (-)-ofloxacin in biological fluids by high-performance liquid chromatography are described. The first method is separation on a chiral stationary phase with bovine serum albumin immobilized on silica gel. The second is the coupling of ofloxacin to L-leucinamide via diphenylphosphinyl chloride activation. The diastereoisomeric derivatives are then separated on a common reversed-phase column. The second method revealed only slight differences in the pharmacokinetics of (+)- and (-)-ofloxacin in humans after an intravenous administration of racemic ofloxacin.