<p>The hydrazide functional group is known for its specific recognition of peroxynitrite. Upon incorporation into rhodamine fluorophores, the resulting fluorescent probes have been widely used for the real-time tracking of peroxynitrite in biological systems. However, the lack of in-depth research on the fundamental reaction mechanism in peroxynitrite detection has limited the optimization of these probes. In this study, we developed two hydrazide-based peroxynitrite probes by linking hydrazine moiety to rhodamine and thio-rhodamine. The responsiveness of these probes toward peroxynitrite was also systematically investigated. Theoretical calculations indicate that the key mechanism of hydrazide-based probes in peroxynitrite detection lies in the reduced Gibbs free energy difference between the ring-open and ring-closed isomers of the oxidized intermediate, which thereby facilitates the ring-opening process. Overall, this study elucidates the reaction mechanism of hydrazide-based peroxynitrite probes from the perspective of Gibbs free energy, providing valuable insights for the rational design and optimization of rhodamine ring-opening probes.</p>