Small molecule screens conducted with living zebrafish have become a commonly practiced technique for small molecule discovery. Embryonic and larval zebrafish exhibit an almost limitless range of phenotypes, from the cellular to the organismal. Consequently, small molecule screens can be designed to discover compounds modifying any of these phenotypes. The compounds discovered by zebrafish screens pose unique challenges for target identification, but the zebrafish also provides several powerful approaches for identifying targets and determining mechanisms of action. Four major approaches have been used successfully, including methods based on comparison of chemical structures, genetic phenocopy, pharmacologic phenocopy and compound affinity. These approaches will continue to facilitate target identification for compounds from zebrafish small molecule screens, and more importantly, to reveal their mechanisms of action.