We validate the utility of ion mobility to measure protein conformational changes induced by the binding of glycosaminoglycan ligands, using the well characterized system of Antithrombin III (ATIII) and Arixtra, a pharmaceutical agent with heparin (Hp) activity. Heparin has been used as a therapeutic anticoagulant drug for several decades through its interaction with ATIII, a serine protease inhibitor that plays a central role in the blood coagulation cascade. This interaction induces conformational changes within ATIII that dramatically enhance the ATIII-mediated inhibition rate. Arixtra is the smallest synthetic Hp containing the specific pentasaccharide sequence required to bind with ATIII. Here we report the first travelling wave ion mobility mass spectrometry (TWIMS) investigation of the conformational changes in ATIII induced by its interaction with Arixtra. Native electrospray ionization mass spectrometry allowed the gentle transfer of the native topology of ATIII and ATIII-Arixtra complex. IM measurements of ATIII and ATIII-Arixtra complex showed a single structure, with well-defined collisional cross section (CCS) values. An average 3.6% increase in CCS of ATIII occurred as a result of its interaction with Arixtra, which agrees closely with the theoretical estimation of the change in CCS based on protein crystal structures. A comparison of the binding behavior of ATIII under both denaturing and non-denaturing conditions confirmed the significance of a folded tertiary structure of ATIII for its biological activity. A Hp oligosaccharide whose structure is similar to Arixtra but missing the 3-O sulfo group on the central glucosamine residue showed a dramatic decrease in binding affinity towards ATIII, but no change in the mobility behavior of the complex, consistent with prior studies that suggested that 3-O sulfation affects the equilibrium constant for binding to ATIII, but not the mode of interaction. In contrast, nonspecific binding by a Hp tetrasaccharide showed more complex mobility behavior, suggesting more promiscuous interactions with ATIII. The effect of collisional activation of ATIII and ATIII-Arixtra complex were also assessed, revealing that the binding of Arixtra provided ATIII with additional stability against unfolding. Overall, our results validate the capability of TWIMS to retain the significant features of the solution structure of a protein-carbohydrate complex so that it can be used to study protein conformational changes induced by the binding of glycosaminoglycan ligands.