ConspectusMethodological development in the fields of genetics, chemical biology, and biochemistry over the last several decades has provided researchers with a diverse set of powerful tools to investigate biological processes. Leveraging these innovations in concert, scientists can now characterize biological pathways at a level of complexity ranging from systems biology down to molecular and atomic detail.Throughout this Account, we illustrate how discoveries made using these tools build on each other to develop a comprehensive understanding of biological pathways. Advancements in genetic sequencing facilitates association of genotypes and phenotypes, independent of biochemical mechanism. Through the biochemical reconstitution of the interactions between biological macromolecules─including the small molecules (ligands and metabolites) and proteins─that participate in these biological pathways, scientists can characterize the specific molecular features that link genotype and phenotype. This facilitates identification of targets within these pathways that can be manipulated to achieve a greater understanding of the biological process or to develop interventions to improve human health outcomes.Specifically, we describe how this toolbox was leveraged to discover and characterize the molecular biochemistry underlying control of pathogenicity in the Gram-positive bacterium