The water oxidation reaction is considered as the bottleneck and rate-determining step in photoelectrochemical water-splitting processes. To achieve efficient water oxidation on the photoanode, sufficient built-in potential on semiconductors under light illumination needs to be maintained. Herein, we introduced the in-situ­ Electric Field Induced Second Harmonic Generation (EFISH) technique to probe the change of built-in potential under the water oxidation reaction process of a single-crystal rutile TiO­₂ photoanode. Under the dark condition, parabolic relation between the second harmonic generation signal and applied potential is observed in TiO₂ depletion region. Under UV illumination, the EFISH signal decreased compared to dark at the photocurrent saturation potential region. Change in the signal is attributed to the decrease of built-in potential in TiO₂. The amount of built-in potential change increased with light illumination and saturated at 520 mV under 15mW/cm² 360 nm UV illumination in an unbuffered solution. This light-induced band edge unpinning effect can be explained by the accumulation of holes at surface states and surface protons at Helmholtz layer that are generated under illumination. Kinetic isotope EFISH experiments suggest that proton-coupled electron transfer (PCET) is the rate-determining step in water oxidation process. Screening of built-in potential under illumination can be mitigated by adding buffer ions, suggesting that the rate-determining step can be altered by changing the solution microenvironment.