Human-based in vitro hepatic experimental systems are now used routinely in drug development. The initial concept of 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 be only assessed in the preclinical phase of drug development using in vitro human-based experimental systems such as human 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. Assay procedures with cryopreserved human hepatocytes using multiwell plates for the evaluation of critical drug properties including metabolic stability, drug-drug interaction potential, and drug toxicity during drug development are described.