► Monoclonal antibodies (mAbs) are essential drugs for treatment of various diseases. ► The heterogeneity of mAbs is significant and should be well characterized. ► Reversed-phase liquid chromatography is a powerful strategy for mAbs analysis. ► Capillary-zone electrophoresis is a good option, because of its MS compatibility. ► Adsorption of mAbs is a major concern with both electrophoresis and chromatography. Recombinant monoclonal antibodies (mAbs) have become particularly relevant for the treatment of autoimmune diseases or cancers. Because of their inherent complexity and for safety reasons, there is a need to develop powerful analytical methods to provide detailed characterizations of mAbs. The aim of the present review is to detail the state-of-the-art of analytical strategies for mAb characterization. It focuses on the most important separation techniques used in this field, specifically, the chromatographic and electrophoretic approaches and their combination with mass spectrometry (MS). Thanks to recent improvements in separation science and MS devices, mAbs can be analyzed more easily. However, there is still a need to find new approaches that avoid adsorption issues.