New sensing materials that are robust, biocompatible, and amenable to array fabrication are vital to the development of novel bioassays. Herein we report the fabrication of ultrathin (ca. 5-8 nm) glass (silicate) layers on top of a gold surface for surface plasmon resonance (SPR) biosensing applications. The nanoglass layers are fabricated by layer-by-layer(LbL) deposition of poly(allylamine) hydrochloride (PAH)and sodium silicate (SiOx), followed by calcination at high temperature. To deposit these layers in a uniform and reproducible manner, we employed a high-volume, low-pressure (HVLP) paint gun technique that offers high precision and better control through pressurized nitrogen gas. The new substrates are stable in solution for a long period of time, and scanning electron microscopy (SEM) images confirm that these films are nearly fracture-free. In addition, atomic force microscopy (AFM) indicates that the surface roughness of the silicate layers is low (rms = 2 to 3 nm), similar to that of bare glass slides. By timing the experimental parameters such as HVLP gun pressure and layers deposited, different surface morphology could be obtained as revealed by fluorescence microscopy and SEM images. To demonstrate the utility of these ultrathin, fracture-free substrates, lipid bilayer membranes composed of phosphorylated derivatives of phosphoinositides (Pis) were deposited on the new substrates for biosensing applications. Fluorescence recovery after photobleaching (FRAP) data indicated that these lipid components in the membranes were highly mobile. Furthermore, interactions of Ptdlns(4,5)P-2 and Ptdins(4)P lipids with their respective binding proteins were detected with high sensitivity by using SPR spectroscopy. This method of glass deposition can be combined with already well-developed surface chemistry for a range of planar glass assay applications, and the process is amenable to automation for mass production of nanometer thick silicate chips in a highly reproducible manner for label-free measurements.