Ion-exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge heterogeneity. The goal of this review is to provide an overview of theoretical and practical aspects of modern IEX applied for the characterization of therapeutic proteins including monoclonal antibodies (Mabs) and antibody drug conjugates (ADCs). The section on method development describes how to select a suitable stationary phase chemistry and dimensions, the mobile phase conditions (pH, nature and concentration of salt), as well as the temperature and flow rate, considering proteins isoelectric point (pI). In addition, both salt-gradient and pH-gradient approaches were critically reviewed and benefits as well as limitations of these two strategies were provided. Finally, several applications, mostly from pharmaceutical industries, illustrate the potential of IEX for the characterization of charge variants of various types of biopharmaceutical products.