The drug discovery and development process requires adequate safety testing for drug toxicity before new drugs can be administered to patients. Hepatocytes are used in vitro to screen compounds for hepatotoxicity, induction of drug-metabolizing enzymes such as cytochrome P450 (P450) isoforms, drug-drug interactions, and establish human relevance for metabolism. Cryopreservation makes it possible to preserve a large quantity of functional hepatocytes. Techniques for cryopreservation of hepatocytes are mainly based on dimethyl sulfoxide (DMSO). However, analyses of metabolic capacities of cryopreserved hepatocytes are often limited by loss of functional integrity of hepatocytes after thawing. Therefore, it is necessary to improve techniques of cryopreservation. We have developed a new cryopreservation technology for mammalian cells based on a wheat protein extract (WPE). We determined whether the WPE can better preserve activities of major P450 isoforms both in suspension and monolayer cultures of hepatocytes. This was achieved by comparing basal and inducible or metabolic activities of isoforms CYP1A1, CYP1A2, CYP2C6, CYP2D2, and CYP3A in rat hepatocytes that were cryopreserved with WPE, relative to fresh cells and those cryopreserved with DMSO. We conclusively show that rat hepatocytes cryopreserved with WPE retain their metabolic competency and their ability to respond to classical P450 inducers when compared with freshly isolated hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for rat hepatocytes. They are an efficient, nontoxic, economic natural product and universal cryoprotectant that is superior to DMSO, which has limitations because of cellular toxicity.