To advance this formate ABS platform from in vitro assays to live-cell imaging applications, we developed coumarin aldehyde fluorophore F-7, which was utilized alongside iridium complex 9 to achieve a robust ratiometric fluorescence response to physiologically relevant concentrations of formate as well as selectivity over potentially competing analytes through iridiumand formate-catalyzed conversion of aldehyde dye F-7 to its alcohol congener F-10. We then established that this formate ABS system is capable of detecting changes in intracellular formate levels with exogenous formate addition. Furthermore, we showed that this ABS platform is sufficiently sensitive to monitor changes in basal formate fluxes in a cell-based model of human colorectal cancer, where genetic knockout of amino acid (serine) and formaldehyde scavenging (ADH5) biochemical pathways for endogenous formate production result in formate deficiency. Future work will explore the application of this twocomponent system for studying contributions of formate as a one-carbon metabolite in cancer and neurodegenerative diseases. In addition to providing a unique chemical tool for deciphering biological contributions of formate, this study provides a starting point for the broader adoption of organometallic chemistry in the design of activity-based sensing probes and related reagents for chemical biology.