Metalloenzymes are critical components of the cellular metal ion pool, catalyzing essential biochemical reactions. We are developing a suite of fluorescence-based probes designed to selectively bind to the active sites of zinc-dependent metalloenzymes, inducing measurable fluorescence changes. These probes enable the detection of metalloenzymes, quantification of metal content within their active sites, and assessment of inhibitor interactions. Historically, the metal ions associated with these enzymes have been considered static and non-labile. However, emerging evidence suggests that, under certain conditions, their metalation state may be dynamic, influenced by fluctuations in local metal ion availability. This presentation will highlight the development of probes targeting key zinc-dependent enzymes, including carbonic anhydrases, metallo-beta-lactamases, and histone deacetylases. Furthermore, we will explore the application of these probes in cellular models of metal dyshomeostasis, providing insights into the dynamic regulation of metalloenzyme activity in biological systems.