PURPOSE: Radiation therapy delivered at ultrafast dose rates, known as FLASH RT, has been shown to provide a therapeutic advantage compared with conventional radiation therapy by selectively protecting normal tissues. Radiochemical depletion of oxygen has been proposed to underpin the FLASH effect; however, experimental validation of this hypothesis has been lacking, in part owing to the inability to measure oxygenation at rates compatible with FLASH. METHODS AND MATERIALS: We present a new variant of the phosphorescence quenching method for tracking oxygen dynamics with rates reaching up to ∼3.3 kHz. Using soluble Oxyphor probes we were able to resolve, both in vitro and in vivo, oxygen dynamics during the time of delivery of proton FLASH. RESULTS: In vitro in solutions containing bovine serum albumin the O depletion g values (moles/L of O depleted per radiation dose, eg, µM/Gy) are higher for conventional irradiation (by ∼13% at 75 µM [O]) than for FLASH, and in the low-oxygen region (<25 µM [O]) they decrease with oxygen concentration. In vivo, depletion of oxygen by a single FLASH is insufficient to achieve severe hypoxia in initially well-oxygenated tissue, and the g values measured appear to correlate with baseline oxygen levels. CONCLUSIONS: The developed method should be instrumental in radiobiological studies, such as studies aimed at unraveling the mechanism of the FLASH effect. The FLASH effect could in part originate from the difference in the oxygen dependencies of the oxygen consumption g values for conventional versus FLASH RT.