AbstractCarboxylesterase 1 (CES1) plays a key role in the metabolism of endogenous biomolecules and xenobiotics including a variety of pharmaceuticals. Despite the established importance of CES1 in drug metabolism, methods to study factors that can vary CES1 activity are limited with only a few suitable for use in live cells. Herein, we report the development of FCP1, a new CES1 specific fluorescent probe with a unique carbonate substrate constructed from commercially available reagents. We show that FCP‐1 can specifically report on endogenous CES1 activity with a robust fluorescence response in live HepG2 cells through studies with inhibitors and genetic knockdowns. Subsequently, we deployed FCP‐1 to develop a live cell fluorescence microscopy‐based approach to identify activity differences between CES1 isoforms. To the best of our knowledge, this is the first application of a fluorescent probe to measure the activity of CES1 sequence variants in live cells.