Affinity-based protein labeling chemical probes are invaluable tools for the selective chemical modification of native proteins. This protein labeling strategy generally relies on "always ON" reactive electrophiles to achieve protein modification. However, reactive electrophiles are also the causes of probe instability and nonselective labeling in complex biological environments. In this paper, we introduce a conditional-activated affinity-based protein labeling strategy in which the probe is activated by peroxynitrite, triggering a labeling reaction with the target protein located in the near proximity. This conditionally activated protein labeling probe was synthesized with a peroxynitrite-responsive acyl hydrazide, a Cy5 fluorescent dye, and a sulfonamide ligand for recognition with human carbonic anhydrase (hCA) protein. We showed that the specific and rapid labeling of hCA can be achieved in the presence of peroxynitrite in living cells and in vivo. Long-term imaging of the target protein in vivo is possible due to the formation of a stable covalent bond. Furthermore, the probe can also be used to detect peroxynitrite secreted from macrophage cells. As compared with the previous reactive oxygen species-activated probes based on the formation of quinone methide intermediate, our hydrazide probe can be easily synthesized and is more resistant to self-hydrolysis in aqueous solutions. We anticipate that such a condition-responsive reagent will become an invaluable tool for research involving the chemical modification of native proteins under oxidative stress conditions.