Monoclonal antibodies (mAbs) are demonstrating major success in various therapeutic areas such as oncology and the treatment of immune disorders. Over the past two decades, novel analytical methodologies allowed to address the chal-lenges of mAbs characterization in the context of their production. However, after administration only their quantification is performed and insights regarding their structural evolution remain limited. For instance, clinical practice has recently highlighted significant inter-patient differences in mAb clearance and unexpected clinical responses, without providing alternative interpretations. Here, we report the development of a novel analytical strategy based on capillary zone electro-phoresis coupled to tandem mass spectrometry (CE-MS/MS) for the simultaneous absolute quantification and structural characterization of infliximab (IFX) in human serum. CE-MS/MS quantification was validated over the range 0.4–25 μg⋅mL-1 corresponding to the IFX therapeutic window and achieved a LOQ of 0.22 μg⋅mL-1 (1.5 nM) while demon-strating outstanding specificity compared to the ELISA assay. CE-MS/MS allowed structural characterization and estima-tion of the relative abundance of the six major N-glycosylations expressed by IFX. In addition, the results allowed charac-terization and determination of the level of modification of post-translational modifications (PTMs) hotspots including deamidation of 4 asparagine and isomerization of 2 aspartate. Concerning N-glycosylation and PTMs, a new normalization strategy was developed to measure the variation of modification levels that occur strictly during the residence time of IFX in the patient’s system, overcoming artefactual modifications induced by sample treatment and/or storage. The CE-MS/MS methodology was applied to the analysis of samples from patients with Crohn’s disease. The data identified a gradual deamidation of a particular asparagine residue located in the complementary determining region that correlated with IFX residence time, while the evolution of IFX concentration showed significant variability among patients.