Molecular orientation-dependent electron transport across supported 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers (SLBs) on semiconducting indium tin oxide (ITO) is reported with an aim towards potential nanobiotechnological applications. A bifunctional strategy is adopted to form symmetric and asymmetric bilayers of DPPC that interact with L-tryptophan, and are analyzed by surface manometry and atomic force microscopy. Polarization-dependent real-time Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS) analysis of these SLBs reveals electrostatic, hydrogen-bonding, and cation–π interactions between the polar head groups of the lipid and the indole side chains. Consequently, a molecular tilt arises from the effective interface dipole, facilitating electron transport across the ITO-anchored SLBs in the presence of an internal Fe(CN)64−/3− redox probe. The incorporation of tryptophan enhances the voltammetric features of the SLBs. The estimated electron-transfer rate constants for symmetric and asymmetric bilayers (ks=2.0×10−2 and 2.8×10−2 s−1) across the two-dimensional (2D) ordered DPPC/tryptophan SLBs are higher compared to pure DPPC SLBs (ks=3.2×10−3 and 3.9×10−3 s−1). In addition, they are molecular tilt-dependent, as it is the case with the standard apparent rate constants , estimated from electrochemical impedance spectroscopy and bipotentiostatic experiments with a Pt ultramicroelectrode. Lower magnitudes of ks and imply that electrochemical reactions across the ITO–SLB electrodes are kinetically limited and consequently governed by electron tunneling across the SLBs. Standard theoretical rate constants accrued upon electron tunneling comply with the potential-independent electron-tunneling coefficient β=0.15 Å−1. Insulator–semiconductor transitions moving from a liquid-expanded to a condensed 2D-phase state of the SLBs are noted, adding a new dimension to their transport behavior. These results highlight the role of tryptophan in expediting electron transfer across lipid bilayer membranes in a cellular environment and can provide potential clues towards patterned lipid nanocomposites and devices.