Drug-induced liver injury(DILI) is one of the major causes of termination of drug development.The establishment of a high-throughput test system to predict potential clinical hepatotoxicity is a valuable approach in the pharmaceutical industry.The high-content imaging-based in vitro assays allow simultaneous detection of cellular multiple parameters in the system.The real-time monitoring of multiple signaling pathways can shed light on many mechanisms of cell injury,with high sensitivity and specificity.Many types of liver cell models have been applied to high-content screening(HCS) so far.This paper introduceds the HCS technology and reviews the data of hepatotoxicity obtained from HCS technology in recent years.At the same time,we discuss the application of this technology in exploring the mechanism of hepatotoxicity and the potential of HCS technology in studying DILI and mechanisms.