Glycosaminoglycans (GAGs) have high negative charge and are biologically and pharmaceutically important because their high charge promotes a strong interaction with many proteins. Due to the inherent heterogeneity of GAGs, multiple oligosaccharides, containing certain common domains, often can interact with clusters of basic amino acid residues on a target protein. The specificity of many GAG-protein interactions remains undiscovered since there is insufficient structural information on the interacting GAGs. Herein, we establish a cluster sequencing strategy to simultaneously deduce all major sequences of the affinity GAG oligosaccharides, leading to a definition of the consensus sequence they share that corresponds to the specific binding domain for the target protein. As a proof of concept, antithrombin III-binding oligosaccharides were examined, resulting in a heptasaccharide domain containing the well-established anticoagulant pentasaccharide sequence. Repeating this approach, a new pentasaccharide domain was discovered corresponding to the heparin motif responsible for binding interferon-γ (IFNγ). Our strategy is fundamentally important for the discovery of saccharide sequences needed in the development of novel GAG-based therapeutics.