Cancer cells exhibit a unique metabolic shift to aerobic glycolysis that has been exploited diagnostically and therapeutically in the clinic. Oncogenes and tumor suppressors alter signaling pathways that lead to alterations of glycolytic flux. Stemming from glycolysis, the hexosamine biosynthetic pathway leads to elevated posttranslational addition of O-linked-β-N-acetylglucosamine (O-GlcNAc) on a diverse population of nuclear and cytosolic proteins, many of which regulate signaling pathways. This unit outlines techniques used to detect metabolic alterations in cancer cells, regulation by signaling pathways, and cellular O-GlcNAcylation.