Combination of surface analytical techniques was employed to investigate the interfacial behavior of the two designed lipids—N-stearoylglycine (1) and its bulky neutral headgroup-containing derivative N-stearoylvaline ethyl ester (2)—at the air–solution interface and as transferred layers on different substrates. Formation of monolayers at the air–water interface was monitored on pure water and on aqueous solutions of different pH. Crystallization effects were visualized at pure water by recording the hystereses in the Langmuir–Blodgett (LB) isotherms and by transferring the layers onto mica, gold (111), and ITO (indium–tin oxide on glass) electrodes. Subphase pH affects the morphology and patch formation in monolayers of 1, as evidenced by BAM measurements. At pH 8.2, formation of well-ordered crystallites is observed, which upon compression elongate according to predominantly 1-D growth mechanism to form a dense layer of crystallites. This effect is not observed in monolayers of 2, whose headgroup is not protonated. The orientation of layers of 1 transferred to the solid supports is also pH dependent, and their stability can be related to formation of a hydrogen-bonded networks. AFM images of 1 exhibited platelets of multilayer phase. The IR spectra of the ITO substrates covered by 1 indicated formation of hydrogen bonds between the amide groups. The nature of the adsorption layer and its organization as a function of potential were studied in-depth by EC STM using Au(111) as the substrate. A model showing the arrangement of hydrogen bonds between adsorbed molecules is presented and related to the observed organization of the layer.