Abstract: Ofloxacin is a new broad-spectrum oral bactericidal antimicrobialagent. Its primary effect is the inhibition of bacterial DNA gyrase. This paper describesthe development of a simple method for its determination using HPLC with UV detection. We used a Waters liquid chromatograph equipped with a Model 490 E multiwave-length detector, a Model 510 pump and a U6K injector .The separation was performed ona Spherisorb C18 column(200 mm ×4.6 mm ID, 5μm)with a mobile phase of mehanol-0.01 mol/L phosphate buffer-0.5 mol/L tetrabutylammonium bromide (35: 65: 4,pH 2.50). The flow-rate was 1.0 ml/min and detection was at 294 nm. A specimen (0. 2 ml) was spiked with the internal standard (norfloxacin) and depro-teinized by adding 1.0ml methanol. The precipitated mixture was shaken and thencentrifuged at 3000×g for 10 min, the supernatant was evaporated at 75°C under a nitro-gen stream. The residue was taken up with 0.4ml of the mobile phase and 50μl aliquotswere injected into the system. The minimal detectable concentration in plasma is 20 ng/ml. There is a linear relation-ship between the peak area ratio over the range of 0.5～4.0 μg/ml with r= 0. 9999.The method has been applied to assay ofloxacin concentration in human plasma. Thepharmacokinetic characteristics were studied.