Ion Exchange Chromatography (IEX) is an efficient and widely employed technique for the comprehensive characterization of therapeutic proteins and peptides. IEX separates biomolecules based on differences in their surface charge arising from amino acid composition and Post- Translational Modifications (PTMs). As a non-denaturing method, IEX is particularly valuable for resolving and quantifying charge variants like deamidation, oxidation, glycosylation, and terminal truncations in monoclonal antibodies, antibody-drug conjugates, and other biotherapeutics. The review provides insights into the principles, advantages, applications, recent advances, and limitations of using IEX (cation and anion exchange modes) for detailed analysis of proteins and peptides.