Understanding how the four-electron oxidation of water to dioxygen proceeds in different materials is critical to the rational design of efficient catalysts towards artificial photosynthetic systems. Here, using in situ electrochemical evanescent wave spectroscopy under oxygen-evolving conditions, we report two intermediates of iridium oxide (IrO_x), which is the most active and stable catalyst characterized to date in acidic medium. The observed potential dependence of the two intermediates indicated that they were associated with different surface sites, and intermediate scavenging experiments using H₂O₂ provided insight into their role during catalysis. Notably, an Ir^V species with an absorption maximum at 450 nm was found to mediate the initial two-electron oxidation of water. Inhibition of the Ir^V species by H₂O₂, combined with computational modeling, indicates that the accumulation and concurrent spin-state change of the Ir^V species is a prerequisite for efficient water oxidation by IrO_x electrodes.