Hyperpolarized γ-Glu-[1-13C]Gly provides a non-invasive means to detect γ-glutamyl transpeptidase (GGT) enzyme activity in vivo with potential for application in functional imaging. Since GGT is most abundant in the proximal tubules of the kidney, and since the properties of γ-Glu-[1-13C]Gly are suitable for in vivo hyperpolarized 13C metabolic analysis, it was proposed as a molecular probe to study kidney function. The aim of the present study is to identify the dose of γ-Glu-[1-13C]Gly that gives high NMR sensitivity in the unsaturated state of the GGT enzyme. Therefore γ-Glu-[1-13C]Gly was polarized with the stable trityl radical OX63 in a custom-designed DNP polarizer (7T, 1.1K) using microwave irradiation at 196.59 GHz and 50 mW. As a first approach to analyze the HP data, method used in [3] was applied. Herein, the reaction rates were calculated by multiplying the kinetic rate constants with the corresponding substrate concentrations, in which the kinetic rate constant is the product of the 13C longitudinal relaxation rate of glycine (~45s) and the ratio of the integrated γ-Glu-[1-13C]Gly and [1-13C]Gly signal amplitude. Benefiting from a narrow spectral linewidth of the hyperpolarized signal (~20 Hz, FWHM), conversion of γ-Glu-[1-13C]Gly to [1-13C]Gly was measurable down to an estimated blood concentration of 32 μM. To address the possibility of substrate saturation of the GGT enzyme in the kidney, different doses of γ-Glu-[1-13C]Gly were administered, corresponding to a blood concentration range of 32 to 500 μM. The variability of the apparent reactions rates between animals is high for all doses of administered γ-Glu-[1-13C]Gly. The rate, however, was proportional with the dose in 7 of 8 rats, and complete saturation of the GGT enzyme cannot be seen in the dosage range tested. This study shows that HP γ-GluGly senses GGT activity with excellent NMR sensitivity and that a broad range of substrate concentrations can be applied to study kidney function. To understand better the distribution of the initial reaction rates and to estimate the dose required to saturate the GGT enzyme, a broader range of substrate doses will be tested, along with simultaneous functional quantification.