Recently, a T₂‐Relaxation‐Under‐Spin‐Tagging (TRUST) MRI technique was developed to quantitatively estimate blood oxygen saturation fraction (Y) via the measurement of pure blood T₂. This technique has shown promise for normalization of fMRI signals, for the assessment of oxygen metabolism, and in studies of cognitive aging and multiple sclerosis. However, a human validation study has not been conducted. In addition, the calibration curve used to convert blood T₂ to Y has not accounted for the effects of hematocrit (Hct). In this study, we first conducted experiments on blood samples under physiologic conditions, and the Carr‐Purcell‐Meiboom‐Gill T₂ was determined for a range of Y and Hct values. The data were fitted to a two‐compartment exchange model to allow the characterization of a three‐dimensional plot that can serve to calibrate the in vivo data. Next, in a validation study in humans, we showed that arterial Y estimated using TRUST MRI was 0.837 ± 0.036 (N=7) during the inhalation of 14% O2, which was in excellent agreement with the gold‐standard Y values of 0.840 ± 0.036 based on Pulse‐Oximetry. These data suggest that the availability of this calibration plot should enhance the applicability of T₂‐Relaxation‐Under‐Spin‐Tagging MRI for noninvasive assessment of cerebral blood oxygenation. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.