The development of new chiral stationary phases has been very important in the accurate analysis of drug enantiomers and their metabolites in biological samples during drug discovery and development. New chiral stationary phases have been developed using conalbumin and flavoprotein from chicken egg whites, which have been applied to a broad range of drug enantiomers. The application and characterization of these two chiral columns for high-performance liquid chromatography have been documented. Both specific and non-specific interactions, based on the silica gel surface and linker moiety, influenced retention and chiral separation of solutes. Interactions between drug enantiomers and proteins, as a pseudo chiral stationary phase, were investigated with affinity capillary electrophoresis, in order to avoid the effects of non-specific interactions. The chiral discrimination region for ketoprofen on the flavoprotein surface was concluded to consist of an α-helix structure. Studies with chemically modified flavoprotein indicated that two types of interactions at the chiral discrimination region were required for chiral separation: a π-π interaction between a tryptophan residue and the aromatic ring of ketoprofen, and an ionic interaction between the carboxyl group of ketoprofen and an amino and carboxyl group of the protein. In the body, drugs and biologically active substances having a carboxyl group have been known to transform various metabolites such as acyl glucuronide. The acyl adenylate has also been noted as a chemically active intermediate of coenzyme A ligation. Both the acyl adenylate and the acyl glucuronide produced protein adducts by reacting with nucleophilic groups such as amino groups on protein molecules. To characterize both active intermediates and protein adducts, analytical techniques conferring highly selective molecular recognition, such as high-performance liquid chromatography and mass spectrometry, were required.