Chemokines play a critical role in inducing chemotaxis, extravasation, and activation of leukocytes both in routine immunosurveillance and autoimmune diseases. Traditionally, to disrupt chemokine function, strategies have focused on blockage of its interaction with the receptor. Recently, it has been demonstrated that binding to glycosaminoglycans (GAGs) is also required for the in vivo activity of many chemokines. Thus, interference with the GAG-binding of chemokines may offer an alternative, valid, anti-inflammatory strategy. However, the potential of using small polyanions to inhibit the interactions between chemokines and cell surface GAGs has not been fully explored. In this study, a mass spectrometry based filtration trapping assay was utilized to study the interactions between two CCR 2 ligands (MCP-1/CCL2 and MCP-3/CCL7) and a series of low molecular weight, polyanionic molecules. Findings were confirmed by using a hydrophobic trapping assay. The results indicated that Arixtra (fondaparinux sodium), sucrose octasulfate, and suramin were specific binders of the chemokines, while cyclodextrin sulfate, although the most highly sulfated molecule among the ones investigated, showed no binding. The binding stoichiometry of the small molecule ligand was determined from the measured molecular weight of the noncovalent complex. Furthermore, the dissociation constant between MCP-3 and Arixtra was determined by using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry, which compared favorably with the result of the isothermal titration calorimetry (ITC) assay. The relative binding affinity of these ligands to MCP-3 was also determined using a competitive filtration trapping assay.