Chemical genomics is a novel approach that allows for the rapid functional analysis of plant proteins, complexes, pathways, and networks. Systematic screens for bioactive small molecules causing specific subcellular phenotypes have been successfully performed in mammalian cells, but thus far, are limited in plants. This protocol describes a systematic chemical screen of plasma membrane recycling markers in plants, using confocal microscopy and the subsequent clustering of subcellular phenotypes, to identify chemicals with desired effects. The method provides an approach to identify novel chemicals for pathway dissection, making chemical genomics more accessible to the scientific community. The matrix of novel chemicals described in this protocol can be expanded and analyzed continuously as more data is collected, increasing our knowledge of the endomembrane system, and accumulating compartment-specific markers and chemical probes that perturb specific aspects of endomembrane trafficking.