As one important category of biological molecules on the cell surface and in the extracellular matrix (ECM), glycosaminoglycans (GAGs) have been widely studied for biomedical applications. With the understanding that the biological functions of GAGs are driven by the complex dynamics of physiological and pathological processes, methodologies are desired to allow the elucidation of cell-GAG interactions with molecular level precision. In this study, a microtiter plate-based system was devised through a new surface modification strategy involving polydopamine (PDA) and GAG molecules functionalized with hydrazide chemical groups. A small library of GAGs including hyaluronic acid (with different molecular weights), heparin, and chondroitin sulfate was successfully immobilized via defined binding sites onto the microtiter plate surface under facile aqueous conditions. The methodology then allowed parallel studies of the GAG-modified surfaces in a high-throughput format. The results show that immobilized GAGs possess distinct properties to mediate protein adsorption, cell adhesion, and inflammatory responses, with each property showing dependence on the type and molecular weight of specific GAG molecules. The PDA-assisted immobilization of hydrazide-functionalized GAGs allows biomimetic attachment of GAG molecules and retains their bioactivity, providing a new methodology to systematically probe fundamental cell-GAG interactions to modulate the bioactivity and biocompatibility of biomaterials.