Automated microscope based High Content Screening (HCS, or HCA, HCI) has gained significant momentum recently due to its ability to study many features simultaneously in complex biology systems. HCS can be used all along the preclinical drug discovery pipeline, it has the power to identify and validate new drug targets or new lead compounds, to predict in vivo toxicity, and to suggest pathways or molecular targets of orphan compounds. HCS also has the potential to be used to support clinical trials, such as companion diagnostics. In this chapter, state of the art HCS approaches are detailed, and challenges specific to HCS are discussed. It should serve as an introduction for new HCS practitioners. More chapters will follow on specific assay examples and on high level informatics analysis.