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Chemically modified biomaterial surfaces (titanium and glass) covered with polyelectrolyte self-assembled films formed by the alternating adsorption of cationic poly-l-lysine (PLL) and anionic poly-l-glutamic acid (PGA) were structurally characterized by atomic force microscopy. Complementary information concerning the thickness and layer-by-layer growth of the films was provided by optical waveguide light-mode spectroscopy. The frequently used ex situ and the rarely used in situ build-up methods were compared. Important aspects of the industrial applicability of these films, their stability in time, and possible differences in their morphology were investigated. The films revealed a granular pattern, with grain diameters of 270 ± 87 nm for glass (up to 8 bilayers) and 303 ± 89 nm for titanium (up to 10 bilayers), independently of the build-up procedure. Both surfaces displayed a rehydration capability, the titanium surface exhibiting a better stability in time. The high roughness values observed at acidic or basic pH are related to the degree of ionization of PGA and PLL.