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, ¹³C magnetic resonance spectroscopy (MRS) allows following the fate of ¹³C-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 ¹³C MRS. In particular, the dynamic ¹³C-labeling of pyruvate and lactate formed from ¹³C-glucose was observed in real time. An ad-hoc synthesis to produce [2,3,4,6,6-²H₅, 3,4-¹³C₂]-D-glucose was developed to improve the ¹³C signal-to-noise ratio as compared to experiments performed following [U-²H₇, U-¹³C]-D-glucose injections. The main advantage of only labeling C3 and C4 positions is the absence of ¹³C-¹³C 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.