Glycosyl-phosphatidylinositol (GPI) anchored proteins have been shown to cluster in lipid-rich microdomains enriched in glycosphingolipids and cholesterol and represent a relatively selective marker of lipid rafts. In recent years several attempts have been made to use fluorescent probes to non-disruptively label these domains in living cells. Here, we transfected endothelial cells with a GPI-anchored thermotolerant green fluorescent protein (ttGFP) to show co-localization of this fluoroprobe with another marker of lipid rafts, urokinase plasminogen activator receptor-1. This fluoroprobe was used to quantify cell surface area occupied by lipid rafts and to examine the effect of various proatherogenic signals on lipid rafts. Exposure of endothelial cells to asymmetric dimethylarginine and oxidized low density lipoprotein (oxLDL), as well as oxidant stress, resulted in the reduction of cell surface area occupied by lipid rafts. Next, the property of ttGFP to undergo a shift in absorbance depending on the clustering of these molecules was utilized to perform proximity imaging (PRIM). Data showed NO-mediated distancing of GPI-ttGFP clustered in lipid-rich microdomains. This unclustering of GPI-anchored ttGFP was not reproduced by pro-oxidant signals and was due to the reduction in membrane-cytoskeleton constrains on the lipid rafts. These findings suggested two fundamentally different mechanisms to modulate lipid rafts: a) the substance regulation of lipid rafts involving the modification of cholesterol and sphingolipids and b) the structural regulation of lipid rafts through disruption of membrane-cytoskeletal interactions switching off the spatial confinement of lipid rafts.