Most of the components of the membrane and protein traffic machinery were discovered by perturbing their functions, either with bioactive compounds or by mutations. However, the mechanisms responsible for exocytic transport vesicle formation at the Golgi and endosomes are still largely unknown. Both the exocytic traffic routes and the signaling pathways that regulate these routes are highly complex and robust, so that defects can be overcome by alternate pathways or mechanisms. A classical yeast genetic screen designed to account for the robustness of the exocytic pathway identified a novel conserved gene, AVL9, which functions in late exocytic transport. We now describe a chemical-genetic version of the mutant screen, in which we performed a high-throughput phenotypic screen of a large compound library and identified novel small-molecule secretory inhibitors. To maximize the number and diversity of our hits, the screen was performed in a pdr5Delta snq2Delta mutant background, which lacks two transporters responsible for pleiotropic drug resistance. However, we found that deletion of both transporters reduced the fitness of our screen strain, whereas the pdr5Delta mutation had a relatively small effect on growth and was also the more important transporter mutation for conferring sensitivity to our hits. In this and similar chemical-genetic yeast screens, using just a single pump mutation might be sufficient for increasing hit diversity while minimizing the physiological effects of transporter mutations.