Drug-induced hepatotoxicity is a major contributor to the high attrition rates of drug candidates and, for the post-launch withdrawals of drugs in the market. The assessment of hepatotoxicity remains difficult because of the challenges associated with in vivo models like poor correlation, ethical issues and high cost. Therefore, alternative in vitro human cells toxicological models are urgently needed. In this study,  human liver cancer cell line (HepG2) spheroids were used as an in vitro model to predict the induced hepatotoxicity of three model drugs (acetaminophen, rifampicin and, ketoconazole). Different concentrations of these drugs were administered after four days of culture to allow the formation of a compact spheroid and, the metabolic activity was determined after 24 hours, which represents the acute toxicity of a drug. The IC50 for acetaminophen was 32.9±6.5 mM, for ketoconazole was 0.22±0.85 mM and, for rifampicin was 37.6±5.3 µM. Therefore, HepG2 cells in a 3D spheroid demonstrated the potential to be in vitro models that mimic the human in vivo situation and allow us to investigate specific parameters in a precisely controlled environment. Authors acknowledge the financial support from Microliver project (PTDC/BBB-ECT/4317/2014) and ComplexiTE project (ERC-2012-ADG 20120216-321266)