Stabilization of surface supported fluid lipid multilayers for underwater characterization is an essential step in making them useful for scalable cell culture applications such as high throughput screening. To this end, we used tetraethyl orthosilicate (TEOS), recently shown to stabilize fluid lipid films while maintaining their fluidity and functionality under water, to stabilize lipid multilayer micropatterns of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The treated multilayers were immersed under water and successfully imaged by atomic force microscopy (AFM), a difficult feat to perform on fluid lipid multilayers without TEOS treatment. The treated lipid multilayer showed an average swelling of approximately 18% in water but remained stable during the imaging process. The TEOS-treated lipid multilayers also proved compatible with cell culture as HeLa, MDCK, and HEK cell types all adhered and grew in high numbers over the multilayers. The results obtained here open the door to the use of fluid lipid multilayers in biotechnology applications such as microarray based high throughput cell assays.