Molecular imaging of biological analytes provides detailed insights into signaling processes. Ratiometric probes are particularly attractive due to the ability to quantify analyte production. However, design strategies for ratiometric probes can be hindered by spectral overlap of the product and reactant species. In this chapter, we provide protocols for the synthesis and application of RF620, a ratiometric probe for H2O2 displaying dramatic changes in both excitation and emission wavelengths, designed using an approach we term chemoselective alteration of fluorophore scaffolds. The probe contains a chemoselective functional group within a fluorescent xanthene scaffold, resulting in the in situ synthesis of a new fluorophore upon reaction with H2O2. Under physiological conditions, RF620 exhibits far-red to near-infrared excitation and emission, and upon reaction with H2O2, RF620 is chemically converted into tetramethylrhodamine, producing a significant (~ 66 nm) blue-shift in excitation and emission. RF620 can be used for ratiometric, molecular imaging of endogenous H2O2 production in living cells.