Self-labeled inhibitors (SLIs) are promising for creating links, ranging from cancer therapy and metastatic pathways to mechanistic elucidation. In this study, a new category of "two-in-one" fluorescent xanthone inhibitors was developed for the systematic evaluation of anticancer activity and the selective imaging of cytoplasm in vitro. These xanthone inhibitors presented high fluorescent brightness, working over a wide pH range enabled by a "switchable reaction" of the heterocyclic backbone. The strength and nature of fluorescence were probed via spectroscopic methods and density functional theory calculations on the molecular level, respectively. Along with the potent anticancer activity, which was demonstrated using MTT and clonogenic assays with high fluorescent brightness in the cytoplasm, SLI 3fd could be established as a modeled self-monitoring drug in cancer therapy.