Glycosphingolipids (GSLs) are amphipathic lipid molecules comprised of a hydrophobic lipid tail and hydrophilic glycan head group. GSLs expressed on cell membranes can alter in response to external stimuli and disease, making them potential markers and/or targets for cellular disease states. Analytical challenges associated with GSL glycan head groups arise from a high degree of compositional similarity. Structural differences in linkage, branching and anomericity must be distinguished from each other, with the added complexity of the presence of isobaric isomers. Analysis using hydrophilic interaction high performance liquid chromatography (HILIC-HPLC) is one of the most common methods for the analysis of released and fluorescently labelled glycans, and provides relative quantitation based on fluorescence detection. The co-elution of structures however can also create ambiguity in structural assignment. The use of ion mobility spectrometry (IMS) as an additional level of separation, based on molecular shape and recorded as collision cross section (CCS), can aid characterisation of isomeric structures. In the work here, we present a workflow for the analysis of procainamide-labelled GSL glycans using HILIC-UPLC-FLR with IMS-MS/MS using a Waters ACQUITY UPLC H-Class and Synapt G2-S. We have created an experimental reference database of GSL glycan standards containing glucose unit (GU), m/z, CCS values and MS/MS spectral profiles for each glycan in all observed ion states. The use of these different attributes in matching experimental data for glycan identification was assessed using in-house developed software. This database was employed in the analysis of genotypically different breast cancer cell lines, in order to identify glycan changes and potential therapeutic targets. Overall, this technology offers a sensitive and robust way of characterising and quantifying glycans which can be applied to the discovery of glycan markers in a range of clinical settings.