The aim of the present investigation was application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the study of antisense oligonucleotides. The influence of several mobile phases, differing with the salt type, their concentration and pH value on the retention and the separation of antisense oligonucleotides has been examined for this purpose. Four different stationary phases were also applied including unmodified silica, silica modified with the use of sulfobetaine groups, polyhydroxy and aminopropyl groups. Such wide range of tested conditions has been useful in better understanding of the retention mechanism of tested compounds. The results obtained during this investigation indicated that greater retention, greater peaks symmetry, as well as more effective separation of oligonucleotides, were obtained for the zwitterionic stationary phase. Moreover, the optimization of tandem mass spectrometry parameters with the use of Central Composite Design was performed and different mobile phases were tested to choose that one, which provided the greatest antisense oligonucleotides peak areas in Multiple Reaction Monitoring mode and consequently, the greatest possible sensitivity. Hydrophilic interaction liquid chromatography was compared with the ion pair chromatography, commonly used in the analysis of oligonucleotides. Both techniques were compared in terms of selectivity of separation as well as the sensitivity of their determination. Obtained results proved that ion pair chromatography provided better results in terms of separation efficiency and peak areas in Multiple Reaction Monitoring for tested conditions. However, these results do not preclude application of hydrophilic interaction liquid chromatography as an alternative chromatographic approach for the oligonucleotides analysis especially when a mobile phase without ion pair reagents is required.