Numerous studies support the fact that a genetically diverse mouse population may be useful as an animal model to understand and predict toxicity in humans. We hypothesized that cultures of hepatocytes obtained from a large panel of inbred mouse strains can produce data indicative of inter-individual differences in in vivo responses to hepato-toxicants. In order to test this hypothesis and establish whether high-throughput in vitro studies using cultured hepatocytes from genetically distinct mouse strains are feasible, we aimed to: (1) determine whether the near-physiological maintenance of the cells isolated from different mouse inbred strains can be achieved, (2) evaluate whether viability and reproducibility of functionality be attained over subsequent isolations and (3) assess the utility of the model for toxicity screening. Our data suggest that cell function and expression of key liver specific genes of hepatocytes isolated from different strains is comparable. These experiments open new opportunities for high-throughput and low-cost in vitro assays that may be used for studies of toxicity in a genetically diverse population.