High-content screening (HCS) has been used in late-stage drug discovery for a decade. In the past few years, technological advances have expanded the role of HCS into the early stages of drug discovery, including high-throughput screening and hit-to-lead studies. More recently, computational advances in image analysis and technological advancements in general cell biology have extended the utility of HCS into target validation and basic biological studies, including RNAi screening. The use of HCS in target validation is expanding the work that can be done at this stage, especially the range of targets that can be characterized, and putting it into a more biological context.