Hydrophobic interaction chromatography (HIC) is a historical strategy used for the analytical purificationand characterization of proteins. Similarly to what can be done in reversed-phase liquid chromatogra-phy (RPLC), HIC is able to separate protein species based on their hydrophobicity, but using differentconditions. Compared to RPLC, the main benefit of HIC is its ability to perform separations under nondenaturing conditions (i.e. physiological pH conditions, ambient mobile phase temperature and no needfor organic solvents) and so an orthogonal method. The goal of this review is to provide a generaloverview of theoretical and practical aspects of modern HIC applied for the characterization of thera-peutic protein biopharmaceuticals including monoclonal antibodies (mAbs), antibody drug conjugates(ADCs) and bispecific antibodies (bsAbs). Therefore, method development approaches, state-of-the-artcolumn technology, applications and future perspectives are described and critically discussed.