A suitable method has been developed and validated for the chiral separation and determination of R,S-mephedrone and one of its metabolites, R,S-4-methylephedrine, and R,S-methylenedioxypyrovalerone (R,S-MDPV) in human hair samples by the in-line coupling between SPE and CD-assisted CE with a previous sample pretreatment procedure based on pressurized liquid extraction. Optimum separation was achieved on a fused silica-capillary of 50 mu m id and 80 cm total length using 12 mg/mL beta-CD in an aqueous solution of 80 mM disodium phosphate at pH 2.5 as the BGE and an applied voltage of 30 kV. The SPE-CE device consists of a short length of a capillary of 2 mm packed with Oasis HLB sorbent, which was inserted near to the inlet end of the CE capillary. Several parameters affecting the in-line preconcentration were evaluated. The LOQs reached for hair samples were 0.05 ng/mg for the enantiomers of mephedrone and its metabolite, and 0.40 ng/mg for the enantiomers of MDPV. The RSDs (%) obtained in intra-and interday studies were less than 10% and the relative recoveries were greater than 80%. The method established in this paper is advantageous for its simplicity, overall analysis time and ability to provide information of both enantiomers of a chiral drug in hair samples.