To separate proteins solely based on their difference in hydrodynamic volume in size exclusion chromatography (SEC), the ionic strength of the mobile phase has to be increased in order to avoid secondary ionic interactions between proteins and the stationary phase. However, adding salts to the mobile phase can have a serious effect on protein aggregation and can lead to artifacts. In the present study, several monoclonal antibodies (mAbs) and the antibody-drug conjugate (ADC), trastuzumab emtansine were selected to study the effect of mobile phase salt additive on aggregation measurements. In a first instance, the same aggregation ratios between the dimeric and monomeric forms of ten mAbs approved by the Food and Drug Administration (FDA) and the European Medicine Agency (EMA) were obtained with three UHP-SEC columns. However, SEC analysis using various amounts of NaCl provided surprising results for rituximab, e.g. presence of 0.8% aggregates with a mobile phase containing 0.2 M NaCl, while no aggregates were observed without NaCl in the mobile phase. Despite the absence of monomeric protein adsorption at the surface of the SEC resin, the comparison of sodium- and potassium-based salts demonstrated the superiority of potassium-based salts to reduce possible secondary electrostatic interactions, mainly between protein dimers and the SEC support as well as to lower protein-salts interaction. To investigate the effect of mobile phase salt additives on SEC measurements, fluorescence spectroscopy provided insights related to the possible contribution of protein tertiary structure. Indeed, biopharmaceuticals could be classified depending on the exposure of their tryptophan residues to the solvent in order to understand their propensity to interact with the stationary phase or/and to undergo self-association.