Hydrophobic interaction chromatography (HIC) is a powerful technique used for purification of biomolecules on both the analytical and preparative scale. This chapter presents an overview of the basic principles of HIC and the different proposed theories for the retention mechanisms as well as the main parameters to consider for the optimization of this chromatographic technique. Additionally, purification strategies and practical aspects are described, providing useful tools for HIC application. Recent selected examples of purification and analysis of different biomolecules by HIC, with particular emphasis on proteins, are also described.