Endomembrane trafficking has a key role for ensuring homeostasis, growth and development, hormonal signaling, and adaptation of eukaryotes to the constantly changing environmental conditions. The complex organization of the endomembrane system implies the need for searching novel tools to specifically probe the regulatory components and dissect the tightly interconnected vesicle transport pathways. Here, we review the large-scale chemical genetic screens, which led to the identification of small molecules with an impact on various parts of the vesicle trafficking network. We discuss the similarities and differences in the organization of the endomembrane systems in yeasts, mammals, and plants based on studies of small molecules and their effects on trafficking hubs, routes, and conserved protein targets.