Abstract: BACKGROUND: Drug discovery and development are predicated on elucidation of the potential mechanisms of action and cellular targets of candidate chemical compounds. Recent advances in high-content imaging techniques allow simultaneous analysis of a range of cellular events. In this study, we propose a novel strategy to identify drug targets by combining genetic screening and high-content imaging in yeast. METHODOLOGY: In this approach, we infer the cellular functions affected by candidate drugs by comparing morphologic changes induced by the compounds with the phenotypes of yeast mutants. CONCLUSIONS: Using this method and four well-characterized reagents, we successfully identified previously known target genes of the compounds as well as other genes involved with functionally related cellular pathways. This is the first demonstration of a genetic high-content assay that can be used to identify drug targets based on morphologic phenotypes of a reference mutant panel.