Drug- induced liver injury is often caused by cytochrome P450- dependent activation of drugs into reactive metabolites. In vitro models, which can mimic in vivo responses and allow the evaluation of initial and adaptive responses to bioactivated compounds over prolonged periods, offer potentially valuable tools for toxicological assessment. We have previously developed a model in which primary hepatocytes (rat, human) are seeded onto ECM-coated domains of optimized dimensions and subsequently cocultivated with murine embryonic fibroblasts (HepatoPac®). This model retains key biochemical functions of in vivo liver with long term stability. Here, we assess the bioactivation and cytotoxicity of acetaminophen (APAP) as a function of culture age in the 96-well HepatoPac format. APAP is a well-known hepatotoxin and exerts its toxic effects through bioactivation associated, in part, with cytochrome P450 3A (CYP3A). Primary rat hepatocytes cultured in the HepatoPac platform were exposed to increasing concentrations of APAP (over 5 days) and assessed for changes in hepatic ATP content, glutathione (GSH) levels, albumin secretion and urea synthesis. Similar concentration-dependent cytotoxicity profiles (AC50=8.4 ± 2.4mM for GSH depletion and 14.17 ± 3.5mM for urea synthesis inhibition) were obtained over the course of the 4-week study. Addition of 200µM L-buthionine (S, R)-sulfoximine (BSO), an inhibitor of GSH synthesis, or 10µM dexamethasone (DEX), an inducer of rat CYP3A1/2, to HepatoPac rat hepatocyte cultures potentiated APAP- induced hepatotoxicity in these cultures irrespective of culture age (over 4 weeks). These findings are consistent with the known in vivo mechanisms of APAP toxicity in rats. In conclusion, rat HepatoPac in a 96-well format provided reproducible APAP-induced cell cytotoxicity profiles over a 4 week period and can be used to assess the effects of chronic exposure to bioactivated compounds.