Comprehensive characterization of physicochemical properties of monoclonal antibodies (mAbs) is a critical process to ensure their quality, efficacy, and safety. For this purpose, mAb analysis at different levels (bottom-up, middle-up) is a common approach that includes rather complex multistep sample preparation (reduction, digestion). To ensure high analysis performance, the development of fully integrated methodologies is highly valuable. Capillary zone electrophoresis is a particularly well-adapted technique for the multistep implementation of analytical strategies from sample preparation to detection. This feature was employed to develop novel integrated methodologies for the analysis of mAb at the middle-up level. Multiple in-line reactions (simultaneous reduction and digestion) were performed for the first time in the separation capillary. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was used as an effective reducing agent under a broad pH range and IdeS (Immunoglobulin degrading enzyme from Streptococcus) as a highly specific enzyme for mAb digestion. Transverse diffusion of laminar flow profile (TDLFP) was applied for reactants mixing. Both in-line sample preparation and separation parameters were optimized under non-denaturing and denaturing conditions. The developed in-line methodologies provided good reproducibility and higher peak efficiencies comparing with off-line assays. They were successfully applied to different mAbs.