Chemical Biology is a strong tool to perform experimental procedures to study the Endomembrane System (ES) in plant biology. In the last few years, several bioactive compounds and their effects upon protein trafficking as well as organelle distribution, identity, and size in plants and yeast have been characterized. Today, several of these chemical tools are widely used to perform mutant screens and establish the trafficking pathway of a given cellular component. This chapter is a guideline to perform multidrug approaches to study the endomembrane system in plant cells. This type of approach is a powerful and useful strategy to thoroughly determine the trafficking of a specific protein as well as to perform mutant screens based on phenotypes produced by drug treatments. On the other hand, a multidrug approach can address the characterization of a new bioactive molecule and find its cellular pathway target. Overall, this approach can unravel mechanisms and identify new players in endomembrane trafficking.