In order to address this question, we combine tethered bilayer lipid membranes (tBLMs) on Au supports with surfaceenhanced infrared absorption (SEIRA) spectroscopy, which was demonstrated to provide insight into the structure, orientation and function of membrane proteins and membrane-active (antimicrobial) peptides. The particular strength of this approach lies in the plasmonic enhancement of IR signals of molecules in close vicinity to the nanostructured Au support (up to ca. 8 nm), which allows selectively detecting processes occurring at the membrane interface or inside the membrane without the interference from the bulk, while changing the ion composition or pH. In this work, we employed the tBLM-SEIRA approach, complemented by density functional theory (DFT), to characterize the effect of different cations on the mechanism of EB incorporation into membranes and provide insight into the structure and nature of EB:cation complexes that are responsible for its ionophore function using calculations. We show that presence of Na + leads to a positive cooperative process of incorporation, while interaction with K + leads to a direct but less efficient insertion into membranes. Furthermore, whereas our SEIRA data shows that membrane-incorporated EB can exchange Na + , K + and Cs + reversibly, the cyclodepsipeptide is (partially) extracted from the membrane by ions according to the series Ca 2+ < Mg 2+ < Li + , providing a rationale for the observed conductivity series. Finally, via comparison of SEIRA and DFTbased spectra we provide an assignment to different EB:cation complexes that, in select cases, contradict those from experiments in organic solvents.