Measurements of Cherenkov emission in tissue during radiation therapy are shown to enable estimation of hemoglobin oxygen saturation non-invasively, through spectral fitting of the spontaneous emissions from the treated tissue. Tissue oxygenation plays a critical role in the efficacy of radiation therapy to kill tumor tissue. Yet in-vivo measurement of this has remained elusive in routine use because of the complexity of oxygen measurement techniques. There is a spectrally broad emission of Cherenkov light that is induced during the time of irradiation, and as this travels through tissue from the point of the radiation deposition, the tissue absorption and scatter impart spectral changes. These changes can be quantified by diffuse spectral fitting of the signal. Thus Cherenkov emission spectroscopy is demonstrated for the first time quantitatively in vitro and qualitatively in vivo, and has potential for real-time online tracking of tissue oxygen during radiation therapy when fully characterized and developed.