A highly selective, sensitive and accurate HPLC method has been developed and validated for the estimation of three fluoroquinolones (FQs) viz., gatifloxacin (GFC), sparfloxacin (SFC) and moxifloxacin (MFC) with 500 microL human plasma using levofloxacin (LFC) as an internal standard (IS). The sample preparation involved simple liquid-liquid extraction of GFC, SFC, MFC and IS from human plasma with ethyl acetate. The resolution of peaks was achieved with phosphate buffer (pH 2.5)-acetonitrile (80:20, v/v) at a flow rate of 1 mL/min on a Kromasil C(18) column. The total chromatographic run time was 18.0 min and the simultaneous elution of GFC, SFC, MFC and IS occurred at approximately 10.8, 12.8, 17.0 and 6.0 min, respectively. The method proved to be accurate and precise at linearity range of 100-10,000 ng/mL with a correlation coefficient (r) of > or =0.999. The limit of quantitation for each of the FQs studied was 100 ng/mL. The intra- and inter-day precision and accuracy values found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of 400 mg GFC tablet.