For their characterization and quality control, monoclonal antibodies are frequently analyzed at the bottom‐up level to generate specific fingerprints that can be used to tackle post‐translational modifications or ensure production consistency between lots. To circumvent time‐consuming and labor‐intensive off‐line sample preparation steps, the implementation of integrated methodologies from sample preparation to separation and detection is highly valuable. In this perspective, capillary zone electrophoresis appears as a choice technique since the capillary can subsequently be used as a vessel for sample preparation and electrophoretic discrimination/detection of the reaction products. Here, a fast in‐line methodology for the routine quality control of mAbs at the bottom‐up level is reported. Simultaneous denaturation and reduction (pretreatment step) were conducted with RapiGest® surfactant and dithiothreitol before in‐line tryptic digestion. Reactant mixing was realized by transverse diffusion of laminar flow profile under controlled temperature. In‐line digestion was carried out with a resistant trypsin to autolysis. The main parameters affecting the digestion efficiency (trypsin concentration and incubation conditions) were optimized to generate mAb electrophoretic profiles free from trypsin interferences. An acidic MS‐compatible BGE was used to obtain high resolution separation of released peptides and in‐line surfactant cleavage. The whole methodology was performed in less than two hours with good repeatability of migration times (RSD = 0.91%, n = 5) and corrected peak areas (RSD = 9.6%, n = 5). CE‐fingerprints were successfully established for different mAbs and an antibody‐drug conjugate.