Tracking the distribution and fate of biomolecules in living cells is crucial to the study of cell biology, and advances in fluorescence microscopy have revolutionized the way biomolecules are monitored in vivo. The discovery that green fluorescent protein could be used to selectively label proteins of interest in vivo via simple genetic encoding enabled researchers to study the spatial and temporal movements of proteins in real time. Over the past decade, several chemical tags have been reported to complement the utility of the fluorescent proteins, and to date, the protein-based tags have demonstrated the highest selectivity in vivo. The Cornish laboratory developed the trimethoprim (TMP)-tag, a chemical tag that exploits the high affinity interaction between TMP and E. coli dihydrofolate reductase (eDHFR) to selectively label proteins with fluorescent probes. Here we present the adaptation of the TMP-tag to enable its use for more demanding live cell imaging experiments. For example, we describe the conversion of the noncovalent TMP-tag into a covalent chemical tag via proximity-induced reactivity to provide a more permanent modification.