Metabolomics is essentially the study of all low molecular weight molecules in a biological system under defined conditions. In glycomics, there is much potential to gain insight into the biosynthesis of novel glycoconjugate structures by probing the metabolome for substrates that are suspected, or known, to be involved in the biosynthetic processes. Recently, we employed the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) in a focused metabolomic study of sugar-nucleotides relevant to the biosynthesis of highly novel carbohydrate modifications on the flagellin of Campylobacter sp. We exploited the unique selectivity of the HILIC-MS method for discriminating between closely related sugar-nucleotide intermediates and allowed their subsequent structural identification using a combination of high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. In addition, the HILIC-MS method permitted screening of selected isogenic mutants for sugar-nucleotide intermediates to determine a role for the corresponding genes on the flagellin glycosylation locus in the biosynthesis of the novel carbohydrate modifications.