The enantiomers of praziquantel, the drug of choice in schistosomiasis, were separated by electrokinetic chromatography with cyclodextrins. Nine anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers. Seven investigated selectors presented chiral interactions with the enantiomers, these cases being interpreted in terms of stability constants and complex mobilities. The best results were delivered by sulfated-β-cyclodextrin, where quasi-equal stability constants were accompanied by extreme selectivity values and was explained on the basis of highly different mobilities of the transient diastereomeric complexes. Since the enantiomer migration order was unfavorable, a simple polarity switch was employed (detection end at anode), which apart from migration order reversal, also resulted in extreme resolution values (Rs > 35) and increased migration times. After optimization (50 mM phosphate buffer pH 2.0, supplied with 15 mM sulfated-β-cyclodextrin, 15 kV, capillary temperature 25°C, short-end injection with 50 mbar × 2 s), analysis time under 10 min were obtained, while still maintaining high resolution (Rs > 10). The method was validated according to the ICH guidelines and application of the method was tested on in-house synthetized R-praziquantel batches and on commercial, combination tablets containing racemic mixture of the drug.