Reversed-phase high-performance liquid chromatography (RP-HPLC) has now become a fundamental part of the set of several analytical techniques used in identifying, isolating, and characterizing therapeutic antibodies. It allows effective separation, accurate characterization, and accurate quantification of antibodies based on their hydrophobic characteristics. Thus, this technique has great potential for evaluating a wide range of physicochemical properties, including the chemical nature of the antibodies, charge variants, and post-translational modifications. RP-HPLC is also extremely useful in the quality control process, ensuring that the antibody preparations are highly pure, consistent, and stable. RP-HPLC is of immense importance in the analysis of antibody fragments, the study of drug conjugation with the antibody, and their stability testing. The latest developments in chromatography include the integration of mass spectrometry and ultra-high-performance chromatography systems, which makes its capability of antibody characterization even greater. The methodology has indeed proven to be crucial in developing biosimilar products, enabling comparative analysis with the reference standard products. As therapeutic antibodies continue to advance in biomedical field, RP-HPLC, together with other analytical techniques, will remain a valuable tool for identification, purification, and characterization of those antibodies so that one can bring safer, more effective antibody-based therapeutic or diagnostic agents from benchside to the patient’s bed.