Three-dimensional (3D) cultured hepatocyte capable of maintaining liver-specific function in an in vivo state over a relatively long period of time have drawn attention as a new method for evaluating the metabolic process, hepatotoxicity and enzyme induction potential of drugs. When human hepatocytes were seeded on a plate for spheroid formation, and cell morphology and albumin secretion were examined, hepatocyte spheroid was stably maintained for at least 21 days after seeding. As a result of drug exposure to this spheroid, sequential metabolic reactions by Phase I and Phase II enzymes and metabolic reactions peculiar to only humans were observed. Moreover, when several drugs were exposed to spheroids and hepatotoxicity was evaluated, stable values were obtained for the 50% inhibitory concentration (IC50) of albumin secretion at 14 and 21 days. The IC50 values of most of the tested drugs were lower than in conventional assays, suggesting that the reported evaluation methods might underestimate hepatotoxicity. Furthermore, examination of mRNA expression level and activity of various cytochrome P450 (CYP) after exposure of typical inducers of CYPs to hepatocyte spheroid resulted in a significant increase in the expression level and activity of each. From these results, it was shown that this 3D hepatocyte spheroid system is suitable for follow-up of metabolic processes, long-term tests of hepatotoxicity and enzyme activity induction potential of drugs.