In the past decade, advances in the field of high-content screening (HCS) have provided researchers with a powerful new screening tool to observe treatment effects on multiple experimental parameters. While extremely useful, HCS has resulted in the collection of large datasets of increased complexity that require intensive analysis. Recently, approaches have been developed to analyze multi-parametric HCS data more completely and, when used in conjunction with RNA interference, target-based biochemistry and structural analysis, these approaches have begun to unlock the potential of this screening format in aiding drug discovery. This review illustrates how the combination of these technologies has been used to successfully drive the drug discovery process.