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Summary Cell surface heparan sulfates modulate many signalling pathways by binding growth factors and growth factor receptors. Expressed in a spatially and temporally regulated manner, these highly sulfated polysaccharides play important roles in all aspects of animal physiology. To understand heparan sulfate-protein binding, it is necessary to develop instrumental sequencing methods. Towards this end, we and others have demonstrated the effectiveness of activated electron dissociation (ExD) tandem mass spectrometry. The value in the ExD approach is that extremely rich tandem mass spectra are produced. The challenge is that bioinformatics methods are needed to convert the raw data into HS saccharide sequences. In this article we describe HS–SEQ, an algorithm developed for this purpose.