Predicting drug-induced hepatotoxicity remains a critical, yet elusive, goal in drug safety studies and pharmaceutical development. This difficulty is, in part, a result of the often-weak relationship between in vitro and animal toxicity models. To address this weakness, we have employed a high-throughput, three-dimensional (3D) cell culture platform containing two cell types to screen a library of 26 small molecule drugs of various mechanisms of action and modes of toxicity. Correlations of in vitro toxicity to in vivo murine toxicity are substantially improved with primary human hepatocytes vs. a human hepatocyte cell line, HepG2. At a murine LD50 (lethal dose for 50% of population) cutoff of 300 mg/kg, the calculated predictivity for primary human hepatocytes is 76%, as compared to a calculated predictivity for HepG2 cells of 54%. These results demonstrate that primary human hepatocytes may be highly predictive of in vivo outcomes, and the use of the 3D chip platform enables substantial reduction in the number of hepatocytes required for in vitro toxicology studies. (c) 2018 American Institute of Chemical Engineers