The use of 2D cell culture has been the mainstay of cell biology and has been applied to all of our modern day drug discovery and development activities. However, the identification of new therapeutics has not achieved the success rates anticipated. One potential explanation is the complexity and lack of predictability of our current research methods, including 2D cell culture. Numerous studies have indicated that cells grown in a flat environment lose critical parameters and thus could provide false information when used in a drug discovery paradigm. In this chapter we describe the advances in 3D cell culture and the potential applications to drug discovery. Highlighted are the differences between cells grown in 2D versus 3D with respect to cell physiology and their responses to external stimuli. These differences can have profound effects on the type of compounds identified and prioritized for further research or development. © 2012 Elsevier Inc.