Isotopic labeling is commonly applied for investigating intracellular metabolism. The general workflow is to first introduce isotopically-labeled metabolites into the culture medium, then at defined time points wash and harvest cells, process samples for metabolomics analysis, and analyze the samples for isotopic enrichment in specified metabolite pools. Here we apply this technique to primary hepatocytes from mice. We introduce either 13C5 glutamine or 13C6 glucose at the typical media concentrations, 1:1 replacing the 12C version with 13C version. Cells are harvested at 0 and 30 min after isotope introduction, metabolites are extracted and then analyzed by GC-MS and LC-MS. The resulting data are used to compare relative 13C isotopic labeling in metabolites between different genetic mutants. This strategy is not suitable for directly quantifying metabolic flux (i.e., Metabolic flux analysis), but is useful for describing relative metabolic flux between two models. The expected time to complete is ~3-5 days.