Cancer progression is accompanied by increases in glucose and glutamine metabolism, providing the carbon and nitrogen required in downstream anabolic pathways. Fructose-6P, glutamine, and acetyl-CoA are central metabolites and substrates of the hexosamine biosynthesis pathway (HBP) to UDP-N-acetylglucosamine (UDP-GlcNAc), an essential high-energy donor for protein glycosylation. Golgi and cytosolic glycosylation pathways are sensitive to UDP-GlcNAc levels, which in turn regulates metabolic homeostasis in a poorly understood manner. To study the hexosamine biosynthesis pathway in cancer cells, we developed a targeted approach for cellular metabolomics profiling by liquid chromatography-tandem mass spectrometry. Human cervical (HeLa) and prostate cancer (PC-3) cell lines were cultured in medium with increasing concentrations of glucose, glutamine, or GlcNAc to perturb the metabolic network. Principal component analysis indicated trends that were further analyzed as individual metabolites and pathways. HeLa cell metabolism was predominantly glycolytic, while PC-3 cells showed a greater dependency on extracellular glutamine. In both cell lines, UDP-GlcNAc levels declined with glucose but not glutamine starvation, whereas glutamine abundance increased UDP-GlcNAc levels 2-3-fold. GlcNAc supplementation increased UDP-GlcNAc 4-8-fold in both HeLa and PC-3 cells. GlcNAc supplementation in HeLa cells induced nonmonotonic changes in NADH/NAD+, NADPH/NADP+, reactive oxygen species, and reduced/oxidized glutathione. In PC-3 cells, GlcNAc supplementation also increased glucose and glutamine uptake and catabolism. Our results suggest that stimulation of the HBP in cancer cells regulates metabolism and redox potential, which might be exploited to target cancer cells.