Glutathione (GSH) is often upregulated in cancer, where it serves to mitigate oxidative stress. gamma-glutamyl-transferase (GGT) is a key enzyme in GSH homeostasis, and compared to normal brain its expression is elevated in tumors, including in primary glioblastoma. GGT is therefore an attractive imaging target for detection of glioblastoma. The goal of our study was to assess the value of hyperpolarized (HP) gamma-glutamyl-[1-C-13]glycine for non-invasive imaging of glioblastoma. Nude rats bearing orthotopic U87 glioblastoma and healthy controls were investigated. Imaging was performed by injecting HP gamma-glutamyl-[1-C-13]glycine and acquiring dynamic C-13 data on a preclinical 3T MR scanner. The signal-to-noise (SNR) ratios of gamma-glutamyl-[1-C-13]glycine and its product [1-C-13]glycine were evaluated. Comparison of control and tumor-bearing rats showed no difference in gamma-glutamyl-[1-C-13]glycine SNR, pointing to similar delivery to tumor and normal brain. In contrast, [1-C-13]glycine SNR was significantly higher in tumor-bearing rats compared to controls, and in tumor regions compared to normal-appearing brain. Importantly, higher [1-C-13]glycine was associated with higher GGT expression and higher GSH levels in tumor tissue compared to normal brain. Collectively, this study demonstrates, to our knowledge for the first time, the feasibility of using HP gamma-glutamyl-[1-C-13]glycine to monitor GGT expression in the brain and thus to detect glioblastoma.