A new reverse phase high performance liquid chromatography (RP-HPLC) method for the quantitative determination of Nimorazole was developed and validated as per ICH guidelines. The analyte was injected into an HIBER C18 column (150 mm × 4.6 mm, 5μm), maintained at ambient temperature and effluent was monitored at 297 nm. The mobile phase consisting of acetonitrile: methanol: buffer (2:3:5 v/v/v). The pH of the mobile phase was adjusted to 4.0 by using O-phosphoric acid. The flow rate was maintained at 1.0 mL/min. and retention time was observed 1.76 min. The developed method shows high specificity for Nimorazole. Calibration curve was plotted with a range from 1-5µg/ml (r2>0.999). The lower limit of quantification (LLOQ) was found to be 0.5μg/ml. The method was validated for parameters like accuracy, precision, recovery, linearity, robustness. This RP-HPLC method is suitable for determining the concentration of Nimorazole and it was applied to routine analysis for determination of the Nimorazole from its formulation during pharmacokinetic study.