Abstract: This study was conducted to establish an in vitro 3D liver model and apply it to the drug liver toxicity evaluation. The 3D multicellular sphere model of HepaRG cells was established by hanging-drop technique for evaluation of liver function. The 3D liver model was used to test the hepatotoxicity of isoniazid and amiodarone hydrochloride compared to the 2D cell culture model. Our results showed that HepaRG cells formed a compact spheriod, and the level of cell albumin, urea and the CYP3A4 activity were significantly higher than that of 2D model. With the treatment of amiodarone hydrochloride in 2D and 3D model, the IC₅₀ were 50 and 100 μmol·L^-1, respectively. When the dose was less than 1 000 μmol·L^-1, isoniazid had no hepatocyte toxicity in 2D model, while the IC₅₀ in 3D model was 700 μmol·L^-1. The LDH activities of both drugs in 3D model showed time-and dose-dependent correlation. The results suggest that this in vitro 3D hanging-drop liver model is good for testing liver functions with a high hepatic drug-metabolizing enzyme activity. Compared with the 2D model, the 3D liver model can accurately evaluate the liver toxicity of drugs. Our results demonstrated the importance of in vitro cell culture models for detection of in vivo-relevant adverse effects of drugs.