Abstract Recently, great interest has been focused on synthetic cathinones since their consumption has increased exponentially. All synthetic cathinones exist as chiral molecules; the biological and/or toxicological properties of cathinones generally differ according to the enantiomers in human body. In this study, a chiral liquid chromatography method was developed to separate and determine the enantiomeric ratio of synthetic cathinones present in “legal highs” acquired in old smart shops or over the Internet. All the synthetic cathinones were efficiently enantio-separated with α and Rs ranging from 1.24 to 3.62 and from 1.24 to 10.52, respectively, using polysaccharide-based chiral stationary phases. All synthetic cathinones, with the exception of 4-methylethcathinone (4-MEC), were present in the commercialized “legal highs” in an enantiomeric proportion of 50:50. One of the studied chiral compounds was 3,4-methylenedioxypyrovalerone (MDPV), one of the most consumed cathinone derivative worldwide. Our research group has recently reported its hepatotoxicity in the racemic form. Thus, the analytical enantioresolution of the MDPV was scaled up to multi-milligram using a semi-preparative amylose tris-3,5-dimethylphenylcarbamate column (20 cm × 7.0 mm ID, 7 µm particle size). Both enantiomers were isolated with high enantiomeric purity (enantiomeric excess > 99 %). The toxicity of S-(−)-MDPV and R-(+)-MDPV was evaluated, for the first time, using primary cultures of rat hepatocytes. It was also possible to verify that MDPV enantiomers showed hepatotoxicity in a concentration-dependent manner, but displayed no enantioselective toxicity in this cell culture model.