Copyright belongs to proceeding publisher ; Glycosaminoglycans (GAGs), being an important constituents of extracellular matrices (ECM), on lipid diffusion in supported lipid bilayers are investigated as a function of concentration. GAGs are attached electrostatically to supported phospholipid (DOPC) bilayers doped with small amounts of cationic lipid (DOTAP) at physiological pH. Lipid dynamics are characterized in terms of the diffusion of fluorescent lipid analogs (DiD/DiO) measured by fluorescence correlation spectroscopy. It is observed that when short chains (≤ 10 disacharide units, LHA) of hyaluronic acid (unsulfated GAG) are bound to the membrane surface, the diffusion coefficient decreases stronger than for medium, MHA, or long chains (≥ 100 disacharide units, HHA). In particular, LHA at micromolar concentration displays a 2-fold increase in Dratio, the ratio between the diffusion coefficients of the lipid fluorescent marker in absence and presence of GAG. At nanomolar concentrations of hyaluronic acid, LHA and HHA behave more or less in a similar manner, i.e. DiD diffusion remains unaltered. Along with the lipid dynamics, we also investigated the impact of GAG on protein dynamics and peptide secondary structure conformation. We observed that protein diffusion becomes slower at the surface of the membrane compared to above the membrane (Figure 2). Similarly, the peptide secondary structure is greatly affected in the presence of sulfated-GAGs (Figure 3). Also, it is observed that hyaluronic acid upon sulfation brings a conformational change in the peptide secondary structure. Diffusion of both heparin binding and non-binding proteins is substantially affected in the presence of heparin in hydrogel matrix (Figure 4).