<p> The development of high-quality screening assays for the identification of biologically active ligands is critical in drug discovery. This thesis is aimed at developing new advances m capillary electrophoresis (CE) for the characterization of the conformational stability and enzymatic activity of protein targets with small molecules. CE provides a convenient platform for unbiased assessment of multiple thermodynamic and kinetic parameters associated with biomolecular interactions involving regulatory protein or isomerase enzymes, where various sample pretreatment steps can be integrated directly in-capillary during analysis. The first two chapters of the thesis (Chapters II, III) outline the development of dynamic ligand exchange-affinity capillary electrophoresis (DLEACE) as a novel strategy for the screening of allosteric ligands based on the differential stability of urea-induced unfolding of various apolholo-protein states of cAMP receptor protein constructs. This work introduced a label-free and multivariate approach for ligand selection based on complementary thermodynamic parameters that allowed for determination of the dissociation constant of protein-ligand interactions over a wide dynamic range (> 10^4, Kd = nM-mM). The subsequent two chapters of the thesis (Chapters IV, V) describe the development of a novel kinetic assay for unbiased characterization of isomerase activity associated with D/L-amino acid metabolism using hydroxyproline epimerase as a model system. Stereoselective resolution of various hydroxyproline isomers was accomplished via off-line or on-line chemical derivatization with dynamic complexation usmg chiral selector(s) in order to screen potential inhibitors for putative epimerase and racemase activity. The integration of both thermodynamic and kinetic strategies for differentiation of mutant from wild-type enzymes was important for revealing the function of a catalytic acid/base cysteine pair in the epimerase active site. Overall, this thesis outlines an integrative framework based on CE for high-quality screening, which is relevant in reducing the high attrition rate of lead candidates in drug development. </p> / Thesis / Doctor of Philosophy (PhD)