Techniques to monitor the oxygen partial pressure (pO 2 ) within implanted tissue-engineered grafts (TEGs) are critically necessary for TEG development, but current methods are invasive and inaccurate. In this study, we developed an accurate and noninvasive technique to monitor TEG pO 2 utilizing proton ( 1 H) or fluorine ( 19 F) magnetic resonance spectroscopy (MRS) relaxometry. The value of the spin-lattice relaxation rate constant (R 1 ) of some biocompatible compounds is sensitive to dissolved oxygen (and temperature), while insensitive to other external factors. Through this physical mechanism, MRS can measure the pO 2 of implanted TEGs. We evaluated six potential MRS pO 2 probes and measured their oxygen and temperature sensitivities and their intrinsic R 1 values at 16.4 T. Acellular TEGs were constructed by emulsifying porcine plasma with perfluoro-15-crown-5-ether, injecting the emulsion into a macroencapsulation device, and cross-linking the plasma with a thrombin solution. A multiparametric calibration equation containing R 1 , pO 2 , and temperature was empirically generated from MRS data and validated with fiber optic (FO) probes in vitro. TEGs were then implanted in a dorsal subcutaneous pocket in a murine model and evaluated with MRS up to 29 days postimplantation. R 1 measurements from the TEGs were converted to pO 2 values using the established calibration equation and these in vivo pO 2 measurements were simultaneously validated with FO probes. Additionally, MRS was used to detect increased pO 2 within implanted TEGs that received supplemental oxygen delivery. Finally, based on a comparison of our MRS data with previously reported data, ultra-high-field (16.4 T) is shown to have an advantage for measuring hypoxia with 19 F MRS. Results from this study show MRS relaxometry to be a precise, accurate, and noninvasive technique to monitor TEG pO 2 in vitro and in vivo.