The amyloid-β peptide is correlated with Alzheimer’s disease and is assumed to cause toxicity by its interaction with the neuron membrane. A custom-made microscope objective based on the supercritical angle technique was developed by our group, which allows investigation of interfacial events by performing surface-sensitive and low-invasive spectroscopy. Applied to Raman spectroscopy, this technique was used to collect information about the structure of polypeptides that interact with a supported lipid bilayer. Notably, the conformation used by amyloid-β(1–40) and amyloid-β(1–42) when interacting directly with or next to the supported lipid bilayer was characterized. We observed two distinct secondary structures, α-helix and β-sheet, which were exhibited by the peptide. These two structures were detected simultaneously. The propensity of the peptide to fold into these structures seemed dependent on both their number of amino acids and their proximity with the supported lipid bilayer. The α-helix structure was observed for amyloid-β(1–42) fragments that were closer to the lipid bilayer. Peptides that were located further away from the bilayer favored the β-sheet structure. Amyloid-β(1–40) was less prone to adopt the α-helix secondary structure.