This unit describes in detail the extraction, purification, and identification of Yersiniabactin the siderophore of Yersinia pestis. Iron is essential for bacterial growth. Although relatively abundant, access to iron is limited in nature by low solubility. This problem is exacerbated for pathogenic bacteria, which must also defeat the host organism's innate defenses, including mechanisms to sequester iron. One solution to these problems is production of water soluble, small molecules with high affinities for iron called siderophores. This protocol has been fine tuned for Yersiniabactin purification but may be easily modified for use in isolating other siderophores or similar molecules.