A simple, rapid, sensitive, specific, accurate, HPLC method was developed and validated as per ICH guidelines for the determination of ofloxacin in eye drop. Thermoseparation products C8 (250 cm × 4.6 mm i.d., 5 μm) column with a mobile phase consisting of Acetonitrile: Buffer in the ratio 35:65 v/v with a flow rate of 1.5 mL/min was used. Detection was carried out at 315 nm using UV detector. Validation parameters were performed to demonstrate system suitability, specificity, precision, linearity and range, accuracy, ruggedness and robustness. The method was linear over the concentration range of 50 – 300 μg/mL. The method showed good recoveries (99.8 – 103.73%) and the relative standard deviations of intra and inter-day assay were 0.554 and 0.677% respectively. The proposed method was found to be precise, accurate, selective and rapid for the determination of ofloxacin in quality control and in assay.