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Abstract Inverse electron demand Diels–Alder reactions betweens‐tetrazines and strained dienophiles have numerous applications in fluorescent labeling of biomolecules. Herein, we investigate the effect of the dienophile on the fluorescence enhancement obtained upon reaction with a tetrazine‐quenched fluorophore and study the possible mechanisms of fluorescence quenching by both the tetrazine and its reaction products. The dihydropyridazine obtained from reaction with a strained cyclooctene shows a residual fluorescence quenching effect, greater than that exerted by the pyridazine arising from reaction with the analogous alkyne. Linear and ultrabroadband two‐dimensional electronic spectroscopy experiments reveal that resonance energy transfer is the mechanism responsible for the fluorescence quenching effect of tetrazines, whereas a mechanism involving more intimate electronic coupling, likely photoinduced electron transfer, is responsible for the quenching effect of the dihydropyridazine. These studies uncover parameters that can be tuned to maximize fluorogenic efficiency in bioconjugation reactions and reveal that strained alkynes are better reaction partners for achieving maximum contrast ratio.