The　rare　N-unsubstituted　glucosamine　(GlcNH3　+)　residues　in　heparan　sulfate　(HS)　have　important　biological　and　pathophysiological　roles.　In　this　study,　a　high-resolution　method　for　the　separation　and　analysis　of　N-unsubstituted　disaccharides　of　heparin/HS　is　described.　Four　N-unsubstituted　disaccharides,　together　with　eight　N-substituted　species,　can　be　well-separated　by　ion-pair　reverse-phase　ultra-performance　liquid　chromatography.　Each　disaccharide　can　then　be　detected　and　its　relative　abundance　quantified　using　electrospray　ionization　mass　spectrometry　in　the　negative　mode.　Because　of　its　high　sensitivity,　without　interference　from　proteins　and　other　sample　impurities,　this　method　is　particularly　useful　in　the　analysis　of　low　content　GlcNH3　+　residues　in　small　amounts　of　biological　materials,　eg.　sera,　tissue　and　cell　culture-derived　samples.　This　would　lead　to　a　better　understanding　of　the　biological　origin　of　GlcNH3　+　residues　and　their　increasingly　important　function　in　human　health　and　disease.　©　2016　Elsevier　B.V.