Substrate hydration is demonstrated to be crucial to film quality during self-assembled (SA) film deposition of tridecafluoro-1,1,2,2,-tetrahydrooetyltrichlorosilane(FOTS) from the vapor phase. The surface hydration was studied by thermogravimetric analysis, and a model was developed to predict the conditions necessary to desorb all of the water adsorbed on a fused silica surface without significantly altering the concentration of the surface hydroxyl groups. The nature of the SA film was investigated as a function of the degree of rehydration of the dehydrated silica surface. The wettability and microstructure of the SA films were examined by water contact angle, ellipsometry, X-ray photoelectron spectroscopy, and atomic force microscopy. There is an optimum degree of substrate hydration, on the order of 1-1.2 monolayers of adsorbed water, required to produce a dense, durable and uniform FOTS film with high water repellency and a smooth surface.