Abstract: Traditional methods in enzyme kinetics analysis have a number of unavoidable limitations. These include the common use of chromophoric reactants as well as coupling enzymes, radioactive materials, high sample consumption, and time consuming analyses, especially for more complicated reaction systems. Current limitations being as they are, using a rapid and widely applicable method to streamline kinetic analyses is the motivation of my research. This thesis describes the development of efficient mass spectrometric assay methods applied to the study of enzyme kinetics and mechanism, reaction intermediate detection, and inhibitor discovery. In this context, the mass spectrometric approach is successfully applied to the characterization of individual enzymes, and is further expanded to address more complicated enzymatic systems, including reaction systems containing multiple competing substrates or multiple enzymes that function in tandem. For the characterization of individual enzymes, a kinetic assay methodology was developed using electrospray ionization mass spectrometry (ESI-MS). Using this assay, the kinetic constants, product inhibition patterns, and catalytic mechanisms for two bacterial carbohydrate sulfotransferases, NodH and Stf0, were rapidly determined. In order to obtain further mechanistic information for the two enzymes, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was employed for the detection of important reaction intermediates. In addition, an immobilized enzyme mass spectrometry (IEMS) technique was used to identify potential inhibitors for NodH. The ESI-MS technique was further developed to analyze multi-substrate parallel reaction systems and evaluate enzyme substrate specificity. Using our multiplex ESI-MS assay, multiple products were quantified simultaneously in one reaction system enabling rapid specificity constant measurements. Specifically, in the multiplex ESI-MS assay developed, the reaction specificity of NodH for four biosynthetic substrates was accurately determined. Finally, the ESI-MS technique was expanded to study multi-enzyme tandem reaction systems. In order to enhance the chemoenzymatic synthesis of novel aminocoumarin antibiotics, an LC-ESI-MS tandem assay was developed and used to monitor tandem incubations of Nov enzymes, including NovL, NovM, and NovP, which are involved in the biosynthetic pathway of the antibiotic novobiocin. The future direction of the research contained in this thesis is to expand the mass spectrometry approach even more to analyze simultaneously multisubstrate multi-enzyme reaction systems.