Abstract: Heparin (Hep) and heparan sulfate (HS) are complex, sulfated GAGs consist of repeating disaccharide units each composed of a uronic acid, either glucuronate or iduronate the latter sometimes sulfated at C-2, i.e. IdoA2S, and a derivative of glucosamine either N-acetylglucosamine, N-sulfated glucosamine, or unsubstituted glucosamine that can be variably O-sulfated. Hep/HS has important biological functions in developmental processes, angiogenesis, blood coagulation, cell adhesion, and tumour metastasis. These involve interactions with a wide variety of proteins, i.e. enzymes, cytokines, growth factors and extracellular matrix proteins through their specific sequences, patterns or densities of sulfation. Mass spectroscopy is now becoming a much more useful tool for the structural analysis of Hep/HS oligosaccharides because of its high detection sensitivity and molecular specificity. MS/MS of heparin/HS oligosaccharides can produce information-rich glycosidic and cross-ring product ions which can be used to determine the sites of acetylation or sulfation. In particular, LC-MS clearly has the potential now to sequence most oligosaccharides, especially with the recent development of a computational framework for dealing with the complex data generated. In this study, porcine intestines heparin was digested by heparinase I. The obtained crude tetrasaccharides (dp4s) were separated and further purified by strong anion exchange HPLC and size-exclusion HPLC. The structures of dp4s were then investigated by the combination of disaccharide analysis, reversed-phase liquid chromatography/electrospray ionization ion trap/time-of-flight mass spectrometry (RP-LC/ESI-IT/TOF MS) and a computational simulation method. The results showed that nine tetrasaccharides (dp4s) were obtained, in which two series of dp4s isomer were existed. The dp4 isomers were successfully separated using IPRP-LC/MS system with hexylamine. One series of dp4s contains △HexA(2S)-GlcNS and △HexA(2S)-GlcNS(6S) disaccharides, and other ones possess △HexA-GlcNS(6S) and △HexA(2S)-GlcNS(6S). Because MSⁿ analysis could produce abundant structurally useful fragments, the former dp4 isomers were selected to investigate fragmentation patterns for the further potential structure analysis by MS. MS² analysis indicated that one dp4 was △HexA(2S)-GlcNS-HexA(2S)-GlcNS(6S) which had one glycosidic cleavage Y₁, two cross-ring cleavages ^0,2A₂ and ^0,3A₂ in the first glucosamine residues (from the non-reducing end) of oligosaccharide. In contrast, the other dp4 was △HexA(2S)-GlcNS(6S)-HexA(2S)-GlcNS which contained two glycosidic cleavages B₂ and Y₁, two cross-ring cleavages ^1,4A₂ and ^0,3A₂ in the first glucosamine residues, and a cross-ring cleavages ^1,5X₄ in the first glucuronic acid residues of the oligosaccharide. This study provides a simple and efficient method for identifying the sequences of the Hep/HS oligosaccharide isomers, which might lead to a greater understanding of the biological roles of Hep/HS oligosaccharides in organisms.