The microbiome and immune system of infants are shaped by various bioactive components of human breastmilk, notably human milk oligosaccharides (HMOs). HMOs represent the third component of breastmilk and exhibit extremely high structural diversity with many isomers. Here, we propose a high throughput and high resolution approach to characterize main oligosaccharides present in breastmilk with high identification level thanks to ion mobility spectrometry. Four pairs of standard HMO isomers, that are (LNT/LNnT), (LNFP I/LNFP V), (3-SL/6-SL) and (2-FL/3-FL), were first investigated under both positive and negative ionization mode using direct introduction-trapped ion mobility spectrometry time-of -flight mass spectrometry (TIMS-TOF). By examining all the ionic species formed (i.e. protonated and deprotonated ions as well as adduct species), every isomer pair could be distinguished through the separation of at least one species, even with a small difference in collision cross section values (as small as 1.5%) thanks to the flexible resolution capacity of the TIMS instrument. Although multiple mobility peaks resulting from different glycan anomeric conformers, open-ring and/or different ionic isomer structures (i.e. various charge site locations), could be observed for some HMO species. The reduction at the reducing-end of HMOs did not significantly facilitate the isomer distinction. Finally, the unambiguous identification of the studied HMOs in a breastmilk sample showed the potential of the approach combining ion mobility separation and MS/MS experiments for high throughput distinction of HMO isomers in complex breastmilk samples without laborious sample preparation.