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The advent of high performance liquid chromatography (HPLC) has greatly accelerated the discovery, purification, and characterization of antimicrobial peptides. Virtually every modern study of an antimicrobial peptide includes or was preceded by a description of its purification. The increased pace of peptide discovery and characterization has resulted from the development of sophisticated column and solvent delivery technology over the last four decades. Interestingly, the modern methods described here derive directly from standard open-column (“low performance”) chromatographic modalities. Therefore, it is not surprising that virtually every method used in traditional column chromatography has been adapted to high performance methods. These include gel filtration, ion-exchange, and reversed-phase chromatography methods described in this chapter.