Alongside IP-RPLC, strong anion exchange chromatography (AEX) and hydrophilic interaction chromatography (HILIC) have been used for impurity profiling of ON products. AEX and HILIC methods have a different separation selectivity compared to IP-RPLC, enabling, besides the separation of shortmer and longmer impurities, the separation of oxidized phosphorothioate (PO) impurities from phosphorothioated FLPs. This difference is related to the difference in acidity (PS has a lower pKa) and hydrophilicity (PS is more hydrophobic) of the PO impurity, which are factors that contribute to the separation of the methods, respectively. However, separation of deaminated products also remains challenging with these LC modes. In the present work, we studied the usefulness of IP-HILIC as an alternative mode for the impurity profiling of phosphorothioated ONs. IP-HILIC has been shown to be highly useful for glycoprotein analysis, where negatively-charged IPRs shield positive charges of the protein backbone, thereby enhancing the contribution of glycosylation to retention and providing unique resolution of protein glycoforms. The application of IP-HILIC to ON analysis has thus far been quite limited and has not been extensively studied. Gong has demonstrated the potential of IP-HILIC to resolve longmer impurities as well as some chemically modified ONs. In this research, we investigated the use of IP-HILIC for the impurity profiling of therapeutic ONs using a representative GalNAc-conjugated, fully phosphorothioated 16-mer ASO (FLP) as model compound. We hypothesized that the addition of positively-charged IPRs to the HILIC eluent reduces the contribution of highly negatively-charged phosphate backbone of ONs on retention, thereby increasing the HILIC selectivity towards the composition of nucleobases and presence of GalNAc-conjugation. We especially aimed for resolving more closely-related impurities comprising the same number of nucleotides as the FLP. For that, we systematically explored the effect of IPR physicochemical properties, hydrophobicity and concentration, column temperature, and eluent pH on ON retention and chromatographic performance. The potential of IP-HILIC for impurity profiling was evaluated by analyzing the shortmer, longmer, PS-PO converted, deaminated and non-conjugated products of the FLP, and applying IP-HILIC-MS to FLP containing low levels of added impurities.