Mitochondrial dysfunction from toxicants is recognized as a causative factor in the development of numerous liver diseases including steatohepatitis, cirrhosis, and cancer. Toxicant-mediated damage to mitochondria result in depressed ATP production, inability to maintain proper cellular calcium homeostasis, and increased reactive oxygen species production. These disruptions contribute to hepatocellular death and lead to liver pathology. Herein, we describe a series of basic and advanced methodologies that can be incorporated into research projects aimed to understand the role of mitochondrial dysfunction in toxicant-induced hepatotoxicity. Protocols are provided for isolation of liver mitochondria, assessment of respiratory function, measurement of mitochondrial calcium uptake, and reactive oxygen species production, as well as characterization of the mitochondrial protein thiol proteome using 2D gel electrophoresis. Data obtained from these methods can be integrated into a logical and mechanistic framework to advance understanding of the role of mitochondrial dysfunction in the pathogenesis of toxicant-induced liver diseases. Copyright © 2010 John Wiley & Sons, Inc.