Hydrophobic interaction chromatography (HIC) is a valuable tool used in protein purification applications. HIC is used in the purification of proteins over a broad range of scales-in both analytical and preparatory scale applications. HIC is used to remove various impurities that may be present in the solution, including undesirable product-related impurities. In particular, HIC is often employed to remove product aggregate species, which possess different hydrophobic properties than the target monomer species and can often be effectively removed using HIC. In this chapter, we provide a description of the basic theory of HIC and how it is used to purify proteins in aqueous-based solutions. Following the theoretical background, the latest in HIC adsorbent technology is described, including a list of commonly used and commercially available adsorbents. The basic procedures for using HIC adsorbents are described next, in order to provide the reader with useful starting points to apply HIC in protein purification applications.