The self-assembly of mixed vesicles of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylserine (PS) phospholipids on top of polyelectrolyte multilayers (PEMs) of poly(allylamine hydrochloride) (PAH), as a polycation, and polystyrenesulfonate (PSS), as a polyanion, is investigated as a function of the vesicle composition by means of the quartz crystal microbalance with dissipation (QCM-D), cryo-transmission electron microscopy (Cryo-TEM), atomic force microscopy (AFM), and atomic force spectroscopy (AFS). Vesicles with molar percentages of PS between 50% and 70% result in the formation of lipid bilayers on top of the PEMs. Vesicles with over 50% of PC or over 80% of PS do not assembly into bilayers. AFS studies performed with a PAH-modified cantilever approaching and retracting from the lipid assemblies reveal that the main interaction between PAH and the lipids takes place through hydrogen bonding between the amine groups of PAH and the carboxylate and phosphate groups of PS and with the phosphate groups of PC. The interaction of PAH with PS is much stronger than with PC. AFS measurements on assemblies with 50% PC and 50% PS revealed similar adhesion forces to pure PS assemblies, but the PAH chains can reorganize much better on the lipids as a consequence of the presence of PC. QCM-D experiments show that vesicles with a lipid composition of 50% PC and 50% PS do not form bilayers if PSS is replaced by alginate (Alg) or poly(acrylic acid) (PAA).