Heparan sulfate (HS) is a sulfated glycosaminoglycan attached to a core protein on the cell surface. Protein binding to cell surface HS is a key regulatory event for many cellular processes such as blood coagulation, cell proliferation, and migration. The concept whereby protein binding to HS is not random but requires a limited number of sulfation patterns is becoming clear. Here we describe a hydrophobic trapping assay for screening a library of heparin hexasaccharides for binders to antithrombin III (ATIII). The hexasaccharide compositions are defined with their building block content in the following format: (DeltaHexA:HexA:GlcN:SO 3:Ac). Of five initial compositions present in the library, (1:2:3:6:1), (1:2:3:7:1), (1:2:3:7:0), (1:2:3:8:0), and (1:2:3:9:0), only two are shown to bind ATIII, namely, (1:2:3:8:0) and (1:2:3:9:0). The use of amide hydrophilic interaction (HILIC) liquid chromatography-mass spectrometry permitted reproducible quantitative analysis of the composition of the initial library as well as that of the binding fraction. The specificity of the hexasaccharides binding ATIII was confirmed by assaying their ability to enhance ATIII-mediated inhibition of Factor Xa in vitro.