Lipid bilayer membranes are complex, dynamic, and functional structures composed of a wide diversity of lipids, proteins, small molecules, and water organized in heterogeneous domains through noncovalent interactions. The structure and motion of these molecules generate large electric fields within the interior of the membrane that are critical to membrane structure and function. Here, we describe how vibrational spectroscopy of unnatural nitrile chromophores places throughout the membrane structure is used to measure electrostatic fields in peptides intercalated in free-standing lipid bilayer membranes of increasing chemical complexity. In combination with electrodynamics simulations, these experiments highlight how common small molecules such as cholesterol dramatically affect membrane structure and dynamics through large changes to membrane electric fields.