The zebrafish is proving to be highly amenable to in vivo small molecule screening. With a growing number of screens successfully completed, a rich interface is being created between disciplines that have historically used zebrafish (e.g., embryology and genetics) and disciplines focused on small molecules (e.g., chemistry and pharmacology). Navigating this interface requires consideration of the unique demands of conducting high-throughput screening in vivo. In this chapter, we discuss design elements of successful zebrafish screens, established screening methods, and approaches for mechanism of action studies following discovery of novel small molecules. These methods are enabling the zebrafish to have an increasingly positive impact on biomedical research and drug development. Copyright © 2011 Elsevier Inc. All rights reserved.