Novel drug design is an essential pillar of medicinal science and pharmaceutical industry. Despite the incredible amount of resources poured into drug discovery, designing a ligand for each newly identified target requires significant investment and time to trial molecular leads. Here I designed an approach to circumvent these challenges in contexts which genetic modification to the molecular target are feasible. Specifically, I use a known molecular scaffold that encompasses an enzyme and high-specificity ligand to modulate the activity of targets as well as image the localization of these targets in both cellular and whole animal settings. This pair includes the bacterial homolog of dihydrofolate reductase (DHFR), in this case, E. coli DHFR (eDHFR) and the corresponding antibiotic, trimethoprim (TMP). This molecular system functions orthogonally to human biology such that the affinity of TMP for eDHFR is >10,000 times more potent than it is for human DHFR. Using eDHFR as a genetically encoded tag inserted either N- or C-terminally to a target gene, the translated product can be targeted with TMP analogs for regulation and imaging applications. I first describe the application of eDHFR as a tag for targeted protein degradation with bifunctional TMP analogs. Secondly, I discuss how endogenous expression of DHFR in bacteria is used for imaging bacterial burden with TMP Positron Emission Tomography (PET) radiotracers. Subsequently, I demonstrate how radiotherapeutic TMP can induce target-specific cell death in engineered human cells expressing eDHFR. Finally, I describe how next generation eDHFR-TMP tools can be developed and exploited for multiplexed PET imaging. Altogether, eDHFR-TMP is a versatile molecular pair that functions as a concise tool for both control and imaging targets in vitro and in vivo.