We describe the resolution of heparan sulfate (HS) isomers by chromatographic methods and their subsequent differentiation by mass spectrometry (MS), ion mobility, and (1)H nuclear magnetic resonance (NMR) analysis. The two purified hexasaccharide isomers produced nearly identical MS spectra, quantitative disaccharide profiles, and partial enzymatic digestions. However, both tandem spectrometry (MS(2)) and ion mobility spectrometry (IMS) indicated structural differences existed. All data suggested the distinction between the two hexasaccharides resided in their uronic acid stereochemistries. Glucuronic (GlcA) and iduronic acids (IdoA) were subsequently defined by (1)H NMR analysis completing the structural analysis and verifying the unique structures initially indicated by MS(2) and IMS. Our results suggest that IMS may be a powerful tool in the rapid differentiation of GlcA and IdoA containing isomers in the absence of prior structural knowledge.