The in vitro metabolisms of 4-bromo-2,5-dimethoxyphenethylamine (2C-B), 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), and 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7) were studied using TESTLIVER (TM)-rat and TESTLIVER (TM)-human, which are new three-dimensional rat and human hepatocyte culture systems, respectively. The metabolites produced in the incubation media were measured by liquid chromatography-tandem mass spectrometry or gas chromatography-mass spectrometry. The data obtained with the in vitro system of rat origin were carefully compared with those obtained by rat in vivo experiments; most of the metabolites found in the in vivo experiments could be reproduced in the present three-dimensional in vitro experiments, although quantitative metabolite distribution patterns obtained with the three-dimensional system was somewhat different from those of in vivo experiments. Because human in vivo experiments are not possible, especially for dubious designer drugs, the in vitro experiments using the three-dimensional human hepatocyte culture system seem very useful for studying human metabolism of new designer drugs as an alternative to human experiments.