Abstract: Considering the importance of carbohydrate moieties on infectivity and host mimicry, there is a need to better understand the biosynthetic pathways of these unusual sugars in order to identify key targets involved in bacterial pathogenesis. Since the in vitro biochemical characterization of the biosynthetic pathway of unusual sugars is currently hindered by the demand for more accurate, sensitive, and rapid analytical methods for characterizing unusual sugar structures, information about the structure and biosynthesis pathway of these compounds is fragmentary. 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 aviable alternative to existing analytical techniques. The objective of this study is to demonstrate the use of electrospray ionization-tandem mass spectrometry (ESI-MS/MS) as a powerful technique for the characterization of enzymatic products in the biosynthetic pathway of deoxythymidine 5’diphosphate-D-rhamnose (dTDP-L-Rha) in E. coliO7. The dTDP-d-glucose-4,6-dehydratase (RmlB), dTDP-4-keto-6-deoxy-glucose-3,5-epimerase (RmlC), and dTDP-4-keto-rhamnose reductase (RmlD) catalyzed reactions were directly monitored by ESI-MS, followed by detailed structural characterization of the final enzymatic products using ESI-MS/MS in the negative-ion mode after minimal cleanup. The biosynthetic pathway of dTDP-L-Rha, beginning from dTDP-L-Rha in three reaction steps catalyzed by RmlB, RmlC, and RmlD, was characterized by ESI-MS/MS. The results obtained were in good agreement with that of traditional high-performance liquid chromatography (HPLC) monitoring and preparation, as well as NMR and ESI-MS structural characterization. Collectively, these data demonstrate that a CID-ESI-MSⁿ based platform is applicable to the facile characterization of the biosynthetic pathway of important unusual dTNP-sugar in the O-chain and offers significant advantages over current methods in terms of speed, sensitivity, reproducibility, automation and reagent costs.