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Glutathione is a crucial component of the redox homeostasis of cells, and altered levels have been linked to human pathologies. We constructed a latent fluorophore (RhoSS) that responds to cellular thiols in vitro and in cyto following intracellular reduction by glutathione to yield rhodamine 110. Importantly, RhoSS was demonstrated to respond to changing levels of glutathione in cells. This compound represents a class of rationally designed latent fluorophores with exciting potential for monitoring cellular thiols.