Two chiral HPLC methods namely method A and method B were developed for the separation of enantiomers of Linezolid. The mobile phases containing hexane, 2-propanol and trifluoro acetic acid (TFA) in the ratio (80:20:0.1, v/v/ v); hexane, ethanol and TFA in the ratio (65:35:0.1, v/v/v) were used in method A and method B, respectively. The assay results of the two methods were checked in terms of F-test variance ratio and found to be less than the table value, confirming their good precision. The enantiomeric separation of Linezolid on different chiral stationary phases was investigated. The two enantiomers of Linezoild were well resolved on a Chiralpak AD, an amylose based stationary phase. Preparative chiral HPLC was carried out to obtain pure (+) enantiomer of Linezolid from its racemate. The method A was extensively validated and found to be robust. The chiral assay of Linezolid in bulk and pharmaceutical formulations (tablet) were found to be 100.4 +/- 0.4 and 101.2 +/- 1.4%, respectively at 95% confidence interval. The percentage recovery of (+) enantiomer (chiral impurity) was found to be 99.2 +/- 1.9 at 95% confidence interval. The limit of detection and limit of quantification of (+) enantiomer were found to be 123 and 374 ng/ml, respectively for 10 microl injection volume.