Human milk oligosaccharides (HMOs) have attracted immense interest in the infant formula industry for their health benefits. Herein, we utilized liquid chromatography-cyclic ion mobility-mass spectrometry (LC-cIM-MS) to develop a robust and multidimensional HMO profiling workflow. This workflow relies on a self-built glycan library, allowing high-throughput searching of oligosaccharides. cIM-MS demonstrated high resolving power in discriminating glycan isomers and increasing peak capacity. This also facilitated the accurate elucidation of most oligosaccharides at sequence levels. A remarkably diverse milk oligosaccharide profile (n = 98) was observed and enabled the discovery of distinctive chromatographic retention patterns. To provide supplementary selectivity for future routine assignment in the absence of standards, we further developed a comprehensive database of experiment-derived traveling wave collision cross section in nitrogen (