We report the study of the interactions of bacterial toxin streptolysin O (SLO) and cholesterol-containing membranes using electrochemical impedance and surface plasmon resonance (SPR) spectroscopy at low hemolytic units on a novel supported membrane interface. The detailed understanding of the process will aid significantly the construction of nanoscale transport channels for biosensing applications. Cholesterol-containing egg PC vesicles, pristine and incubated with SLO toxin, were fused onto a hexyl thioctate (HT)-modified gold substrate. The charge-transfer resistance of the resulting lipid membrane, which is related to the formation of the transmembrane pores, is measured with the aid of an electroactive probe. Impedance spectra were collected over a range of 0.1-100 kHz, and the obtained complex resistance was fit to an equivalent circuit. The charge-transfer resistance decreases for increasing SLO concentration, following a first-order exponential decay. The two-part membrane interface was further characterized with SPR spectroscopy. For the hexyl thioctate support layer, an equivalent monolayer thickness of 1.3 nm was determined. This value suggests a loosely packed structure of the monolayer on gold, presenting an ideal platform for permeability studies. A comparative study on the fusion behavior of vesicles with and without SLO induced pores revealed no substantial difference for the two systems, indicating that the pore formation has no adverse effect on the integrity of the vesicles. The resulting lipid membrane thickness from pre-perforated lipids was found to be 3.2 nm, suggesting that one leaflet is knocked off during the fusion process and a hybrid membrane is formed. A slightly higher thickness value of 3.4 nm was obtained for membranes from non-perforated vesicles. Deposition of lipids and subsequent incubation with SLO, as monitored by SPR, shows that the HT surface chemistry allows partial insertion of the toxin into the membrane, indicating unique properties as compared to the previously explored long-chain alkylthiols.