Objective: To develop and validate a simple and rapid reversed phase high performance liquid chromatographic (RP-HPLC) method for the determination of Gemifloxacin (GFX) in human urine.Methods: GFX was isolated from urine samples after acidification using methylene chloride. Good chromatographic separation was achieved using C18 Ultrasphere (250 mm × 4.6 mm, 5 µm.) analytical column maintained at 25 °C. The mobile phase consisted of methanol and 0.1 M phosphate buffer pH 3 in the ratio of (48: 52, v/v), respectively. The analysis time was 10 min at a 1.0 ml/min flow rate. The UV detection was carried out at 272 nm.Results: GFX has been eluted at 7.5 min. Linearity was obtained over a concentration range of 20-200 ng/ml (r2>0.999). The extraction recovery of GFX from urine samples was 60%. The proposed method demonstrated excellent intra-and inter-day precision and accuracy within 1.19% and 100.65 %, respectively. The limit of detection (LOD) was found to be 6.3 ng/ml.Conclusion: Simple and accurate RP-HPLC method for determination of GFX in human urine was developed and validated. The method was successfully applied for determination of GFX in human urine samples from healthy volunteers up to 24 hours after oral administration of 320 mg gemi floxacin tablets.Â