AbstractThe hexosamine biosynthesis pathway (HBP) is a glucose metabolism pathway that produces uridine diphosphate N‐acetyl glucosamine (UDP‐GlcNAc). Substantial changes in HBP, including elevated HBP flux and UDP‐GlcNAc levels, are associated with cancer pathogenesis. Particularly, cancer cells expressing oncogenic Kirsten rat sarcoma virus (KRAS) are highly dependent on HBP for growth and survival. To differentiate between HBP metabolites in KRAS wild‐type (WT) and mutant (MT) lung cancer cells, a simultaneous quantitative method for analyzing seven HPB metabolites was developed using ultra‐high‐performance liquid chromatography‐tandem mass spectrometry. A simple method without complicated preparation steps, such as derivatization or isotope labeling, was optimized for the simultaneous analysis of highly hydrophilic HBP metabolites, and the developed method was successfully verified. The intra‐ and inter‐day coefficients of variation were less than 15% for all HBP metabolites, and the recovery was 89.67–114.5%. All results of the validation list were in accordance with ICM M10 guidelines. Through this method, HBP metabolites in lung cancer cells were accurately quantified, and it was confirmed that all HBP metabolites were upregulated in KRAS MT cells compared with KRAS WT lung cancer cells. We expect that this will be a useful tool for metabolic research on cancer and for the development of new drugs for cancer treatment.