Human-based in vitro hepatic experimental systems are now used routinely in drug development. The rationale for the use of human-based in vitro systems is based on the known species-species differences in drug properties. Human-specific drug properties, by definition, cannot be defined using nonhuman experimental animals, and therefore can only be assessed in the preclinical phase of drug development using in vitro human-based approaches. A widely applied human-based in vitro experimental system for preclinical evaluation of drug properties is human hepatocytes. Our laboratory was one of the first to successfully isolate highly viable and functional hepatocytes from human livers, and we recently developed cryopreservation procedures to retain high viability and high attachment efficiency of the isolated hepatocytes. Successful cryopreservation of human hepatocytes greatly enhances the utility of this valuable in vitro experimental system, allowing storage, transport, convenient scheduling of experimentation and repeat experimentation using hepatocytes isolated from the same donors. Effective assays have been developed with cryopreserved human hepatocytes using multiwell plates for the evaluation of critical drug properties, including metabolic stability, drug-drug interaction potential, and drug toxicity. Human hepatocytes represent an alternative experimental system that plays a significant role in the 3Rs - the reduction, refinement, and replacement of the use of animals in preclinical drug development research.