In this work, we aimed to explore the structural changes of Aβ during the early stages of the lag-phase before aggregation to macroscopic fibrils. In particular, we asked the question, if re-insertion of Aβ into the lipid membrane as specific membrane-associated structures affects the misfolding trajectory. Towards this goal, we use the combination of tethered bilayer lipid membrane (tBLM) systems and surfaceenhanced infrared absorption (SEIRA) spectroscopy, which has become an important spectroscopic tool to monitor a broad range of membrane-confined processes ranging from activity of antimicrobial peptides to the folding and function of membrane proteins. The infrared (IR) absorption fingerprint of peptides and proteins is highly sensitive to the secondary structure (in both type and composition) via collective vibrations of the backbone peptide bonds, the socalled amide modes. SEIRA spectroscopy further benefits from a plasmonic enhancement in the vicinity of the Au support (up to approx. 10 nm; Figure ), such that processes at or in the tBLM are detected selectively and contributions from the bulk are avoided. Furthermore, the surface-selection rule results in a preferential IR absorption by vibrational modes with dipole changes along the surface normal, allowing to quantify the orientation of α-helices or β-sheets within the membrane. In order to specifically address the capacity of Aβ to (re)insert into membranes, we adapted a strategy that we used previously for the study of antimicrobial peptide-membrane interactions. Accordingly, we designed (a) solid-supported lipid monolayers on top of dense lipid tethers and (b) bilayer lipid membranes with free-floating bilayer fragments, such that Aβ-membrane incorporation is prevented or can occur, respectively (Figure ). Monitoring the time-dependent evolution of SEIRA signals due to Aβ-membrane interactions, we find that the ability to penetrate the membrane interior promotes the transition from anti-parallel to parallel β-sheet structures, and thus potentially the formation of toxic species interfering with the membrane integrity. Furthermore, analyzing the spectral changes using the density function theory (DFT)-derived orientation dependence of the polarized SEIRA spectra, we quantify the average orientation of the βsheet strands when associated with the membrane, providing an additional experimental observable for comparisons to structures proposed in the literature.