High content screening (HCS) has emerged as an important platform technology for early drug discovery from target identification through in vitro ADME/Tox. The focus is now on implementing multiplexed assays, developing and using advanced reagents and developing and harnessing more sophisticated informatics tools. Multiplexed HCS assays have the potential to dramatically improve the early drug discovery process by creating systems cell biology profiles on the activities of compounds. It is predicted that multiplexed HCS assays will accelerate the overall workflow and produce deeper functional knowledge, thereby permitting better decisions on what compounds to pursue.