The mammalian brain relies primarily on glucose as a fuel to meet its high metabolic demand. Among the various techniques used to study cerebral metabolism, ¹³ C magnetic resonance spectroscopy (MRS) allows following the fate of ¹³ C-enriched substrates through metabolic pathways. We herein demonstrate that it is possible to measure cerebral glucose metabolism in vivo with sub-second time resolution using hyperpolarized ¹³ C MRS. In particular, the dynamic ¹³ C-labeling of pyruvate and lactate formed from ¹³ C-glucose was observed in real time. An ad-hoc synthesis to produce [2,3,4,6,6- ² H ₅ , 3,4- ¹³ C ₂ ]-D-glucose was developed to improve the ¹³ C signal-to-noise ratio as compared to experiments performed following [U- ² H ₇ , U- ¹³ C]-D-glucose injections. The main advantage of only labeling C3 and C4 positions is the absence of ¹³ C- ¹³ C coupling in all downstream metabolic products after glucose is split into 3-carbon intermediates by aldolase. This unique method allows direct detection of glycolysis in vivo in the healthy brain in a noninvasive manner.