A rapid and sensitive reverse phase high performance liquid chromatographic (RP-HPLC) method for the analysis of levofloxacin from bulk materials, dosage formulations and human serum is described. This isocratic method employs, a Nucleosil, C18 (10 microm, 25 cm x 0.46 cm) column with a mobile phase of water and acetonitrile (6:5), where in phosphoric acid was used to adjust the pH to 2.9 and propylparaben as an internal standard. Optimization of levofloxacin analysis was carried out using multivariate calibration technique and detector response was recorded at five different wave lengths. A linear response (r > 0.9999) was observed in the range of 40 to 10000 ng ml(-1). The method shows good recoveries, intra and inter-day relative standard deviations were less than 1.2 %. Validation parameters as specificity, accuracy and robustness were also determined. The method can conveniently be used for analysis of levofloxacin pharmacokinetic levels in human serum and pharmaceutical formulations.