A key to the development of lipid membrane-based devices is a fundamental understanding of how the molecular structure of the lipid bilayer membrane is influenced by the type of lipids used to build the membrane. This is particularly important when membrane proteins are included in these devices since the precise lipid environment affects the ability to incorporate membrane proteins and their functionality. Here, we used neutron reflectometry to investigate the structure of tethered bilayer lipid membranes and to characterize the incorporation of the NhaA sodium proton exchanger in the bilayer. The lipid membranes were composed of two lipids, dioleoyl phosphatidylcholine and cardiolipin, and were adsorbed on gold and silicon substrates using two different tethering architectures based on functionalized oligoethylene glycol molecules of different lengths. In all of the investigated samples, the addition of cardiolipin caused distinct structural rearrangement including crowding of ethylene glycol groups of the tethering molecules in the inner head region and a thinning of the lipid tail region. The incorporation of NhaA in the tethered bilayers following two different protocols is quantified, and the way protein incorporation modulates the structural properties of these membranes is detailed.