A new HPLC method for the determination of Metronidazole was established. The determination was performed by using a Kinetex C18 analytical column with a gradient mobile phase system consisting of 0.05M KH₂PO4 (pH 3.5), pH adjusted with ortho phosphoric acid (mobile phase A) and Acetonitrile (mobile phase B). The flow rate was 1ml/min. and quantitation was achieved with UV detection at 230 nm, based on peak area. The retention time obtained was 5.35 ± 0.004 and the method was validated according to ICH guidelines regarding linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, robustness, and selectivity. Excellent linearity was observed for the calibration curve with an excellent correlation coefficient 0.9999. Limit of detection was 0.78 μg mL−1; limit of quantitation 2.37 μg mL−1. The developed method was found to be accurate and sensitive and is ideally suited for analysis of MTZ in pure form, pharmaceutical dosage form and spiked urine samples.