A novel economic procedure for stereoselective separation and determination of R(+) - and S(-)- ropivacaine was described using different thin layer chromatographic plates and different cyclodextrins at different temperatures. The spots were detected either with iodine vapors or UV lamp 254nm, followed by densitometric measurements at 262nm. Comparative study was achieved using different cyclodextrins namely, hydroxypropyl-β-cyclodextrin (HP-β-CD), methyl-β-cyclodextrin (M-β-CD), and Dimethyl-β-Cyclodextrin (DM-β-CD) as chiral selectors. The mobile phase enabling successful resolution of (±) ropivacaine was acetonitrile: water (17:3 v/v) containing 1mM of DM-β-CD at ambient temperature 25±20C. All variables affecting the resolution, such as concentration of different chiral selectors, temperature, and pH were investigated and the conditions were optimized. The procedure provided a linear response over the concentration range of 1.25 - 35 μg/spot for determination of R (+)- and S- (-)enantiomers (r = 0.9998, n = 8), (r = 0.9998, n = 6) with acceptable precision (% RSD < 1.5) and accuracy (% RE= -1.18 to 2.00). Limits of detection and quantification were found to be 0.29μg/spot and 0.96 μg/spot for -(R) and 0.26μg/spot and 0.86μg/spot for -(S) respectively. The developed method was validated and proved to be robust. Ropivacaine sample solution was found to be stable for one weak in methanol. The proposed method was found to be selective and accurate for identification and quantitative determination of enantiomeric purity of ropivacaine in bulk powder and pharmaceutical dosage form.