This study focused on the use of IP-HILIC-MS for the separation and assignment of impurities of phosphorothioated ONs comprising the same number of nucleotides as the FLP. We systematically studied the influence of IP-HILIC separation parameters, such as type and concentration of IPR, eluent pH and column temperature, using a poly(dT) ladder and a model FLP (GalNAc-conjugated PS 16-mer ASO) with several of its related impurities (N-1, N+1, PO, DA and NC). We showed that IPRs in the HILIC eluent (i) reduce the relative contribution of the highly polar phosphate moieties to ON retention, (ii) alter the HILIC separation selectivity inducing resolution of DA, PO and NC impurities from the FLP while maintaining separation of N-1 and N+1 from the FLP, and (iii) reduces peak widths of phosphorothioated ONs, most probably due to reduction of diastereomer separation. Optimal resolution for the model FLP and its related impurities was obtained using an IP-HILIC eluent of pH 6.3 with 25 mM TEA, and a column temperature of 80 °C. The feasibility of the optimized method for impurity profiling was indicated by IP-HILIC-MS analysis of the model FLP containing several impurities at the 2% level. Comparison with current IP-RPLC, AEX, and HILIC methods showed that only IP-HILIC was able to resolve the DA product -an essential but difficult to detect impurity -from the FLP. Moreover, the selectivity and sensitivity IP-HILIC-MS showed suitable of revealing a large number of minor impurities in an unpurified standard ON. Therefore, we believe that IP-HILIC-MS shows interesting potential for impurity profiling of therapeutic ONs.