We have developed a means of producing thin, oriented lipid monolayers which are stable under repeated washing and which may be useful in biosensing or surface- coating applications. Phosphotidylcholine (PC) and the glycosphingolipid (GSL) GM1 were used as representative lipids for this process. Initially, a mixed self-assembled monolayer of octanethiol and hexadecanethiol was constructed on a clean gold surface. This hydrophobic surface was then brought into contact with a thin lipid layer that had been deposited at the air/liquid interface of a solution by evaporating a mixture of lipid in hexane on top of a layer of water. The lipid layer, now deposited on the gold surface, was then heated to cause intercalation of the fatty acid and alkanethiol chains, and cooled to form a highly stable film which withstood repeated rinsing and solution exposure. Presence and stability of the film was confirmed via ellipsometry, FTIR, and QCM, with an average overall thickness of ∼3.5 nm. These films may be potentially useful in biotoxin detection or as a protein resistant layer for the prevention of biofouling.