Glutamine is a major substrate for biosynthesis. It contributes to multiple pathways required for cell proliferation, supports antioxidant defense via glutathione synthesis, and sustains the tricarboxylic acid (TCA) cycle through anaplerosis. Glutamine-fueled anaplerosis and related biosynthesis can be studied in detail in melanoma using stable isotope (¹³C) labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis of metabolite amounts and labeling. Detailed protocols for the assay of polar metabolites (including amino acids, TCA cycle, and glycolysis metabolites) and fatty acids by these methods following cell treatment with ¹³C-glutamine or ¹³C-glucose are presented.