To separate proteins solely based on their difference in hydrodynamic volume in size exclusion chro-matography (SEC), the ionic strength of the mobile phase has to be increased in order to avoid secondaryionic interactions between proteins and the stationary phase. However, adding salts to the mobile phasecan have a serious effect on protein aggregation and can lead to artifacts. In the present study, several mon-oclonal antibodies (mAbs) and the antibody-drug conjugate (ADC), trastuzumab emtansine were selectedto study the effect of mobile phase salt additive on aggregation measurements. In a first instance, the sameaggregation ratios between the dimeric and monomeric forms of ten mAbs approved by the Food andDrug Administration (FDA) and the European Medicine Agency (EMA) were obtained with three UHP-SECcolumns. 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 wereobserved without NaCl in the mobile phase. Despite the absence of monomeric protein adsorption at thesurface of the SEC resin, the comparison of sodium- and potassium-based salts demonstrated the supe-riority of potassium-based salts to reduce possible secondary electrostatic interactions, mainly betweenprotein dimers and the SEC support as well as to lower protein-salts interaction. To investigate the effectof mobile phase salt additives on SEC measurements, fluorescence spectroscopy provided insights relatedto the possible contribution of protein tertiary structure. Indeed, biopharmaceuticals could be classifieddepending on the exposure of their tryptophan residues to the solvent in order to understand theirpropensity to interact with the stationary phase or/and to undergo self-association.