The composition of biological lipid bilayer membrane creates a complex structural and electrostatic environment that regulates important membrane functions. The alignment of molecular dipole moments from lipid head groups and water molecules located at membrane-water interface creates the dipole potential (Vd). The dipole field (Fd) generated from this potential is the largest and about 1-10 MV/cm in magnitude, determined by indirect measurement techniques. It is located entirely within the membrane interior, and therefore, direct measurement and manipulation of Fd is a challenge. Here, we present direct measurement of dipole electrostatic field within vesicular bilayer using vibrational Stark effect (VSE) shifts of nitrile oscillator in response to the local electrostatic field. We intercalated four different α-helix transmembrane peptides containing unnatural amino acid, p-cyanophenylalanine (p-CN-Phe) at four unique positions into unilamellar vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Molecular Dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the head-group region of the lipid and one buried in the interior of the bilayer were performed to examine the structure of nitrile with respect to the membrane normal, the assumed direction of Fd, by quantifying both tilt of the helix in bilayer and conformational rotation of the p-CN-Phe side chain at steady-state. As nitrile systematically moved towards the membrane interior, the vibrational absorption energies of nitrile showed blue shift. We used the measured VSE shifts and nitrile orientations within the membrane and calculated the magnitude of Fd to be 8 −11 MV/cm, within the range reported in the literature. We increased the chemical and structural complexity of model membrane by adding cholesterol into vesicle composition at different concentrations and determined that Fd changes as function of membrane composition and temperature.