Federal regulations concerning the safety and efficacy of biopharmaceuticals require the implementation of a comprehensive toolbox of physicochemical and biological characterization methods. In order to demonstrate consistent overall structure, even minute differences in primary structure and post‑translational modifications (PTMs) have to be detectable in therapeutic proteins. Because of their remarkable capability of revealing small changes in molecular structure, high performance liquid chromatography (HPLC) and mass spectrometry (MS) rate among the most powerful technologies for comprehensive protein analysis. This article details the potential of both methods with regard to revealing methionine oxidation, a chemical modification that may be induced during downstream processing and storage of biopharmaceuticals. The benefits and limitations of bottom-up, middle-down, and top‑down HPLC–MS analysis will be demonstrated for the detection of oxidation variants in a therapeutic monoclonal antibody (mAb).