Mutant IDH leads to 2HG production, which drives glioma development. 13C MRS monitoring of hyperpolarized [1-13C]α-ketoglutarate (αKG) metabolism to 2HG and glutamate provides a non-invasive assessment of the IDH mutation and normal metabolism, respectively. However, monitoring 2HG production in vivo is challenging because its resonance is within 0.1 ppm of the natural abundance [5-13C]αKG signal of the [1-13C]αKG substrate. Here, we utilized [1-13C-5-12C]αKG, which eliminated the [5-13C]αKG peak. This new approach, combined with an optimized sequence, made it possible to readily monitor the production of both 2HG and glutamate in a patient-derived glioma animal model and in normal brain.