The goal of this study was to better understand the chromatographic conditions in which monoclonalantibodies (mAbs) of broad hydrophobicity scale and a cysteine conjugated antibody-drug conjugate(ADCs), namely brentuximab-vedotin, could denaturate. For this purpose, some experiments were car-ried out in HIC conditions using various organic modifier in natures and proportions, different mobilephase temperatures and also different pHs. Indeed, improper analytical conditions in hydrophobic inter-action chromatography (HIC) may create reversed-phase (RP) like harsh conditions and therefore proteindenaturation. In terms of organic solvents, acetonitrile (ACN) and isopropanol (IPA) were tested with pro-portions ranging from 0 to 40%. It appeared that IPA was a less denaturating solvent than ACN, but shouldbe used in a reasonable range (10–15%). Temperature should also be kept reasonable (below 40◦C), tolimit denaturation under HIC conditions. However, the combined increase of temperature and organiccontent induced denaturation of protein biopharmaceuticals in all cases. Indeed, above 30–40◦C and10–15% organic modifier in mobile phase B, heavy chain (HC) and light chain (LC) fragments dissociated.Mobile phase pH was also particularly critical and denaturation was significant even under moderatelyacidic conditions (pH of 5.4).Today, HIC is widely used for measuring drug-to-antibody ratio (DAR) of ADCs, which is a critical qualityattribute of such samples. Here, we demonstrated that the estimation of average DAR can be dependenton the amount of organic modifier in the mobile phase under HIC conditions, due to the better recoveryof the most hydrophobic proteins in presence of organic solvent (IPA). So, special care should be takenwhen measuring the average DAR of ADCs in HIC.