Abstract: Novel gram-negative bacteria have lipopolysaccharides terminating in repeating oligosaccharides which comprise the O-antigen. The glycosyltransferases (GTs) assembling the O-chain utilize lipid-linked acceptor substrates and nucleotide sugar donor substrates. However, the lack of a rapid and simple method for monitoring glycosyltransferase activity preclude the elucidation of the function of putative GTs. Mass spectrometry is a rapid, sensitive, and accurate approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides a viable alternative to existing analytical techniques. In this study, a simple and efficient assay for glycosyltransferase activity by matrix-assisted laser desorptionionization (MALDI) mass spectrometry with collision-induced dissociation (CID) was demonstrated by wabD (in E. coli O77 O-antigen gene cluste) enzymatic reaction, wfgD (in E. coli O152 O-antigen gene cluste) enzymatic reaction and wfeD (in S. boydii O14 O-antigen gene cluste) enzymatic reaction, respectively. The rapid and direct detection of the enzymatic reaction was achieved by subjecting a small amount (0.3 μL) of the reaction mixture to MS analysis without chromatographic separation or desalting steps, and subsequent MS-MS analyses of the product via collision-induced dissociation enabled the structures of products of enzyme-catalyzed reactions to be determined. Collectively, these data demonstrate that CID MALDI-TOF MS based platform is applicable to the facile determination of the enzymatic activity of other newly cloned glycosyltransferases and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility, automation and reagent costs.