Conversion of acetoacetate (AcAc) to β-hydroxybutyrate (β-HB) by the mitochondrial enzyme β-hydroxybutyrate dehydrogenase depends upon NADH availability. Previous studies have shown the potential of β-HB-to-AcAc ratio to reflect the redox state in rat hearts and lymphoma cells. Here, we assessed the value of HP [1,3-13C]-AcAc in brain. We demonstrated the potential to probe AcAc to β-HB conversion in normal mice and mice with glioblastoma. Significantly higher levels of [1-13C]-β-AcAc and [1-13C]-β-HB were observed in tumor-bearing mice compared to control mice. Consistent with lower levels of NADH measured in tumors, the [1-13C]-β-HB-to-[1-13C]-β-AcAc ratio trended towards a decrease compared to normal brain.