Abstract The characterization of the impurities of pharmaceutical monoclonal antibodies (mAbs) is crucial for their function and safety. Capillary zone electrophoresis (CZE) is one of the most efficient tools to separate charge variants of mAbs; however, peak characterization remains difficult, since the hereby used background electrolytes (BGEs) are not compatible with electrospray ionization‐mass spectrometry (ESI‐MS). Here, a method that allows the separation of intact mAb charge variants is presented using CZE‐ESI‐MS, combining a cationic capillary coating and an acidic BGE. Therefore, a successive multiple ionic‐polymer layer coating was developed based on diethylaminoethyl‐dextran–poly(sodium styrene sulfonate). This coating leads to a relatively low reversed electroosmotic flow (EOF) with an absolute mobility slightly higher than that of antibodies, enabling the separation of variants with slightly different mobilities. The potential of the coating is demonstrated using USP mAb003, where it was possible to separate C‐terminal lysine variants from the main form, as well as several acidic variants and monoglycosylated mAb forms. The presented CZE‐MS method can be applied to separate charge variants of a range of other antibodies such as infliximab, NISTmAB (Reference Material from the National Institute of Standards and Technology), adalimumab, and trastuzumab, demonstrating the general applicability for the separation of proteoforms of mAbs.