The interplay between glycolysis and gluconeogenesis is central to carbohydrate metabolism. Here, we describe novel methods to assess carbohydrate metabolism using [ 13 C]-probes derived from glycerate, a molecule whose metabolic fate in mammals remains underexplored. Isotope-based studies were conducted via NMR and mass spectrometry analyses of freeze-clamped liver tissue extracts after [2,3- 13 C 2 ]glycerate infusion. The ex vivo investigations were correlated with in vivo measurements using hyperpolarized [1- 13 C]glycerate. Application of [ 13 C]glycerate to N-nitrosodiethylamine (DEN)-treated rats provided further assessments of intermediary carbohydrate metabolism in hepatocellular carcinoma. This method afforded direct analyses of control versus DEN tissues, and altered ratios of 13 C metabolic products as well as unique glycolysis intermediates were observed in the DEN liver/tumor. Isotopomer studies showed increased glycerate uptake and altered carbohydrate metabolism in the DEN rats.