Ultrathin (<20 nm) polypeptide multilayer films are assembled by the electrostatic adsorption of alternating monolayers of poly(L-lysine) and poly(L-glutamic acid) onto carboxylic acid terminated alkanethiol-modified gold surfaces. These polypeptide multilayer films are hydrophilic, can bind electroactive anions such as ferri/ferrocyanide, and are stable when immersed in organic solvents such as 1,2-dichloroethane (1,2-DCE). A combination of ex situ polarization-modulation Fourier transform infrared reflection-absorption spectroscopy (PM-FTIRRAS) and surface plasmon resonance (SPR) measurements is used to characterize the film deposition and the incorporation of D2O and electroactive ions. Electrochemical cycling of the polypeptide films in 1,2-DCE is used to reversibly oxidize the ferrocyanide ions in the film, and in situ PM-FTIRRAS measurements demonstrate that more than 95% of the ferrocyanide ions can be converted to ferricyanide without loss to the organic phase. These ultrathin films will be used to study both ion and electron transport across the film/1,2-DCE interface.