A new spirocyclized rhodol-based fluorescent probe has been developed for detecting mitochondrial Cu⁺. Alkylation of the hydroxy group of a xanthene moiety with a tris(2-pyridylmethyl)amine-based ligand induced the formation of a non-fluorescent spirocyclic structure. The reaction with Cu⁺ in the presence of submillimolar concentrations of glutathione at physiological pH resulted in the elimination of the ligand together with an increase in the fluorescence of the rhodol fluorophore. This probe was used to visualize mitochondrial Cu⁺ in copper supplemented cells.