Kinetic isotope effect (KIE) measurements are a powerful tool to interrogate the microscopic steps in enzyme catalyzed reactions and can provide detailed information about transition state structures. However, the application of KIE measurements to study enzymatic reactions is not widely applied due to the tedious and complex analytical workflows required to measure KIEs with sufficient precision. In this thesis I described the development of a novel competitive KIE measurement method using MALDI-TOF-MS and the investigation of the transition state of glycosyltransferase enzyme BshA from B. subtilis. We developed a method for the direct measurement of competitive KIEs using a whole molecule matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). This approach enabled quantitative measurements of both relative isotope abundance of an analyte and fractional conversion F in single measurements without the need for purification prior to analysis. The application of this MALDI-TOF MS approach has demonstrated the precision of KIE measurements comparable to those obtained using competitive radioisotope labelling, and NMR based approaches while requiring smaller amounts of stable isotope labelled substrates. Using two chemoenzymatic approaches, we then synthesized 5 substrates for the application of our method to investigate the transition state of BshA: UDP-GlcNAc (3.1), [1''-13C]UDP-GlcNAc (3.2), [2''-13C]UDP-GlcNAc (3.3), [13C6]UDP-GlcNAc (3.4) and [2''-2H]UDP-GlcNAc (3.5). Finally, we have begun to work on the synthesis of [1''-18O]UDP-GlcNAc and describe an approach to prepare this substrate that is currently underway in the lab. Application of the quantitative whole molecule MALDI-TOF MS approach enabled us to determine multiple competitive KIEs for the enzymatic reaction catalyzed by BshA. While previous studies suggested a front-face SNi (DNAN) TS for the conjugation of UDP-GlcNAc and L-malate, our KIE results show that a stepwise mechanism resulting in the formation of a discrete, though likely short lived, oxocarbenium ion intermediate is more likely. Our method be applied to study other glycosyltransferases whose mechanisms still remain to be elucidated and to design TS based inhibitors for enzymes involved in different bacterial infections. Future work on automation of this method would simplify the KIE measurement process and increase reproducibility making the measurement of KIEs for TS analysis a more experimentally accessible technique for the broader enzymology research community.