Small molecule inhibitors provide powerful tools to characterize highly dynamic and complex eukaryotic cell pathways such as those mediating membrane traffic. However, a lack of easy and generalizable assays has constrained identification of novel inhibitors despite availability of diverse chemical libraries. Here, we report a facile growth-based strategy in yeast to screen for pathway-specific inhibitors. The approach uses well characterized synthetic genetic growth defects to guide design of cells genetically sensitized for inhibition of chosen pathways. With this strategy, we identified a family of piperazinyl phenylethanone compounds as inhibitors of traffic between the trans-Golgi network (TGN) and endosomes that depends on the clathrin adaptor complex AP-1. The compounds did not significantly alter other trafficking pathways involving the TGN or endosomes, indicating specificity. Compound treatment also altered localization of AP-1 in mammalian cells. These previously uncharacterized inhibitors will be useful for future studies of clathrin-mediated transport in yeast, and potentially in other organisms. Furthermore, the easily automated technology should be adaptable for identification of inhibitors of other cellular processes.