Chemical genetic screening can be described as a discovery approach in which chemicals are assayed for their effects on a defined biological system. The zebrafish, Danio rerio, is a well-characterized and genetically tractable vertebrate model organism that produces large numbers of rapidly developing embryos that develop externally. These characteristics allow for flexible, rapid and scalable chemical screen design using the zebrafish. We describe a protocol for screening compounds from a chemical library for effects on early zebrafish development using an automated in situ based read-out. As screenings are carried out in the context of a complete, developing organism, this approach allows for a more comprehensive analysis of the range of a chemical's effects than that provided by, for example, a cell culture-based or in vitro biochemical assay. Using a 24-h chemical treatment, one can complete a round of screening in 6 d.