A simple, rapid, specific, precise and accurate NP-HPLC method has been developed and validated for the Separation of enantiomers of crizotinib. The chromatographic separation was achieved on chiralcel OD-H (25 × 0.46 cm, 5 μ) column at a detector wavelength of 268 nm using an isocratic mobile phase comprising a mixture of n-hexane-isopropyl alcohol-methanol- diethyl amine (40:30:30:0.5 v/v/v/v), pumped at a flow rate of 1.0 ml/min. S & R enantiomers, eluted at the retention times of 4.9 and 6.1 min respectively. The method was validated with respect to parameters such as specificity, linearity, precision, robustness, limit of detection, limit of quantification, system suitability. The developed method was found to be suitable for separation and quantification of enantiomers of crizotinib.