The　rare　N-unsubstituted　glucosamine　(GlcNH　3　+　)　residues　in　heparan　sulfate　(HS)　have　important　biological　and　pathophysiological　roles.　Because　of　their　low　natural　abundance,　the　use　of　chemically　generated,　structurally　defined,　N-unsubstituted　heparin/HS　oligosaccharides　can　greatly　contribute　to　the　investigation　of　their　natural　role　in　HS.　However,　the　sequencing　of　mixtures　of　chemically　generated　oligosaccharides　presents　major　challenges　due　to　the　difficulties　in　separating　isomers　and　the　available　detection　methods.　In　this　study,　we　developed　and　validated　a　simple　and　sensitive　method　for　the　sequence　analysis　of　N-unsubstituted　heparin/HS　oligosaccharides.　This　protocol　involves　pH　4　nitrous　acid　(HNO　2　)　degradation,　size-exclusion　HPLC　and　ion-pair　reversed-phase　liquid　chromatography-ion　trap/time-of-flight　mass　spectrometry　(IPRP-LC-ITTOF　MS).　We　unexpectedly　found　that　absorbance　at　232　nm　(normally　used　for　specific　detection　of　C4-C5　unsaturated　oligosaccharides)　was,　in　most　cases,　still　sufficiently　sensitive　to　also　simultaneously　detect　saturated　oligosaccharides　during　HPLC,　thus　simplifying　the　positional　analysis　of　GlcNH　3　+　residues.　The　IPRP-LC-ITTOF　MS　system　can　supply　further　structural　information　leading　to　full　sequence　determination　of　the　original　oligosaccharide.　This　new　methodology　has　been　used　to　separate　and　sequence　a　variety　of　chemically　generated,　N-unsubstituted　dp6　species　containing　between　1　and　3　GlcNH　3　+　residues　per　oligosaccharide　in　different　positional　combinations.　This　strategy　offers　possibilities　for　the　sequencing　of　natural　N-unsubstituted　oligosaccharides　from　HS　and　should　also　be　applicable,　with　minor　modification,　for　sequencing　at　N-sulfated　residues　using　alternative　pH　1.5　HNO　2　scission.　©　2015　The　Author　2015.　Published　by　Oxford　University　Press.　All　rights　reserved.