Liver is considered to be one of the most important organs in evaluating drug toxicity because it is functionally interposed between the absorption site and the systemic circulation and is a major site of metabolism and elimination of xenobiotics, which make it a preferred target for drug toxicity. Drug-induced liver injury (DILI) rarely occurs but is the leading cause in acute liver failures that necessitate organ transplants and safety recalls of successfully launched drugs from the market. Although liver toxicity can be triggered by drugs through different mechanisms, mitochondrial dysfunction is considered to be one of the major mechanisms for DILI. Drugs could prompt the necrosis/apoptosis of hepatocytes by preventing mitochondrial energy production and releasing mitochondrial pro-apoptotic proteins into the cytoplasm. Therefore, if high-throughput in vitro prediction systems to detect mitochondrial dysfunction could be developed to identify hepatotoxic drugs with reasonable specificity in the early stages of drug development, it would undoubtedly improve the drug development process and impact the human safety. In this symposium, we will present some case studies for evaluating drug-induced mitochondrial toxicity, including the in vitro screening system developed in our laboratory using rat primary hepatocytes to evaluate the mitochondrial function in the early stages of drug development.