Supported planar lipid bilayers (SLBs) prepared by spreading of unilamellar vesicles on hydrophilic substrates such as silicon dioxide are frequently used to investigate lipid−protein interactions by means of surface-sensitive methods. In recent years, the receptor lipid phosphatidylinositol-4,5-bisphosphate (PtdIns-[4,5]P 2 ) became particularly important as a significant number of proteins bind to this lipid at the inner leaflet of the plasma membrane. Here, we investigated how the lipid PtdIns[4,5]P 2 distributes between the two leaflets of an SLB on SiO 2 surfaces. We prepared SLBs on SiO 2 by spreading small unilamellar vesicles and quantified the adsorption of PtdIns[4,5]P 2 binding proteins providing information about the accessibility of PtdIns[4,5]P 2 . We compared protein binding to PtdIns[4,5]P 2 in SLBs with that in lipid monolayers on a 1,1,1-trimethyl-N-(trimethylsilyl)silanamine-functionalized SiO 2 surface using reflectometric interference spectroscopy and atomic force microscopy. Our results clearly demonstrate that the accessibility of PtdIns[4,5]P 2 for protein binding is reduced in SLBs compared to that in supported hybrid membranes, which is discussed in terms of PtdIns[4,5]P 2 distribution in the two leaflets of SLBs.