The aim of the current review is to summarize present methods used for the determination of reactive metabolites, which can predict drug-induced liver injury (DILI) in drug discovery and development. DILI is one of the most frequent reasons for the withdrawal of an approved drug from the market, and it accounts for up to 50% of acute liver failure cases. This review is structured into three sections. The first section is a general overview of the relationship between drug metabolism and liver injury. The second section introduces in vitro methods for the assessment of reactive metabolites for drug discovery and development. In the third section, limitations and future directions for the development of methods for predicting DILI are described.