Oligosaccharides have multiple functions essential for health. Derived from the condensation of two to several monosaccharides, they are structurally diverse with many co-occurring structural isomer families, which makes their characterization difficult. Thanks to its ability to separate small molecules based on their mass, size, shape and charge, ion mobility-mass spectrometry (IM-MS) has emerged as a powerful tool for separating glycan isomers. Here, the potential of such a technique for the rapid characterization of main human milk oligosaccharides (HMOs) was investigated. Our study focused on eighteen HMO standards. The IM-MS analysis enabled to distinguish almost all the HMOs studied, in particular thanks to the single ion mobility monitoring (SIM2) acquisition using the trapped ion mobility spectrometry (TIMS) device, providing high ion mobility resolution and enhanced ion mobility separation. Alternatively, the combination of IM-MS separation with MS/MS experiments has proven to increase performance in identifying HMOs and especially isomers poorly separated by ion mobility alone. Finally, collision cross-section (CCS) values are provided for each species generated from the eighteen HMOs standards, which can serve as an additional identifier to characterize HMOs.