A rapid, sensitive and selective hydrophilic interaction liquid chromatography–tandem mass spectrometric (HILIC–MS/MS) method for the determination of levofloxacin in human plasma was developed. Levofloxacin and ciprofloxacin (internal standard) were extracted from human plasma with dichloromethane and analyzed on an Atlantis HILIC Silica column with the mobile phase of acetonitrile-ammonium formate (100 mM, pH 6.5) (82:18 v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r > 0.999) over the concentration range of 10.0–5000 ng/ml. The lower limit of quantification for levofloxacin was 10.0 ng/ml using 20 μl plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four QC levels were 2.9–7.8% and −7.3% to −2.2%, respectively. The recoveries of levofloxacin and ciprofloxacin were 55.2% and 77.3%, respectively. This method was successfully applied to the pharmacokinetic study of levofloxacin in humans.