Hydrogen sulfide (H 2S) is an integral signaling molecule in biology with complex generation, translocation, and metabolism processes that are intertwined with cellular thiols. Differentiating the complex interplay between H 2S and biological thiols, however, remains challenging due to the difficulty of monitoring H 2S and thiol levels simultaneously in complex redox environments. As a step toward unraveling the complexities of H 2S and thiols in sulfur redox homeostasis, we present a dual-fluorophore fragmentation strategy that allows for the ratiometric determination of relative H 2S and cysteine (Cys) or homocysteine (Hcy) concentrations, two important metabolites in H 2S biosynthesis. The key design principle is based on a nitrobenzofurazan-coumarin ( NBD-Coum) construct, which fragments into spectroscopically differentiable products upon nucleophilic aromatic substitution with either H 2S or Cys/Hcy. Measurement of the ratio of fluorescence intensities from coumarin and the NBD-Cys or NBD-Hcy adducts generates a sigmoidal response with a dynamic range of 3 orders of magnitude. The developed scaffold displays a rapid response (<1 min) and is selective for sulfhydryl-containing nucleophiles over other reactive sulfur, oxygen, and nitrogen species, including alcohol- and amine-functionalized amino acids, polyatomic anionic sulfur species, NO, and HNO. Additionally, NBD-Coum is demonstrated to differentiate and report on different oxidative stress stimuli in simulated sulfur pools containing H 2S, Cys, and cystine.