Hydrophilic interaction liquid chromatography (HILIC) coupled to drift tube ion mobility spectrometry (DTIMS) was used to separate diastereomers of five-unit oligonucleotides containing 0, 1, 2 or 3 phosphorothioate (PS) linkages. Multiplexed DTIMS (where ions are pulsed into the drift tube according to a pre-encoded sequence) and post-acquisition processing using an innovative demultiplexing tool were investigated. The electric field inside the drift tube was optimized to achieve the highest resolving power.