Liver damage is a common issue of synthetic cathinones abuse. Human stem cell-derived hepatocyte-like cells (HLCs) has been suggested to hepatotoxicity studies, by their ability to maintain hepatic-specific phenotype. Furthermore, all cathinone derivatives are chiral, and their biological effects can differ for each enantiomer. Thus, the aim of this work was to evaluate the cytotoxicity and metabolism of pentedrone and methylone enantiomers using HLCs models. Human neonatal mesenchymal stem cells were differentiated into HLCs by a three-step differentiation protocol and maintained under 2D and 3D culture conditions. Subsequently, pentedrone and methylone enantiomers were isolated by HPLC using a chiral stationary phase. Cell viability was evaluated through CellTiter-Glo assay and the formation of methylone and pentedrone metabolites was analysed by GS-MS. Racemates of pentedrone and methylone exhibited potential hepatotoxic in a concentration-dependent manner in both models. It was also observe a different cytotoxic profile for pentedrone enantiomers in HLCs 3D, being R-(-)-pentedrone the most cytotoxic. Concerning HLCs 2D metabolic assays, S-(-)-methylone was preferentially metabolized via N-demethylation, whereas the R-(+)-methylone by O-demethylation and N-hydroxylation. Although, in HLCs 3D, R-(+)-methylone was preferential metabolized by all metabolic pathways, except for O-demethylation. Regarding pentedrone enantiomers, metabolic pathways studied were more pronounced for R-(-)-pentedrone, namely N-demethylation and β-keto reduction in both models. Overall, this study revealed stereoselectivity on cytotoxicity and metabolism pathways for pentedrone and methylone.