Protein-based liquid chromatography stationary phases are used in bioaffinity chromatography for studying drug-protein interactions， the determination of binding affinities， competitive and allosteric interactions， as well as for studying protein-protein interactions. This review addresses the development and characterization of protein-based stationary phase， and the application of these phases using frontal and zonal chromatography techniques. The approach will be illustrated using immobilized heat shock protein 90α and the immobilized estrogen related receptor stationary phases. In addition， the review discusses the use of the protein-coated magnetic beads for ligand and protein fishing as well as for the identification of unknown ligands from cellular or botanical extracts.