Protein-membrane interactions play an important role in various biological phenomena, such as material transport, demyelinating diseases, and antimicrobial activity. We combined vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy with theoretical (e.g., molecular dynamics and neural networks) and polarization experimental (e.g., linear dichroism and fluorescence anisotropy) methods to characterize the membrane interaction mechanisms of three soluble proteins (or peptides). α₁-Acid glycoprotein has the drug-binding ability, but the combination of VUVCD and neural-network method revealed that the membrane interaction causes the extension of helix in the N-terminal region, which reduces the binding ability. Myelin basic protein (MBP) is an essential component of the myelin sheath with a multi-layered structure. Molecular dynamics simulations using a VUVCD-guided system showed that MBP forms two amphiphilic and three non-amphiphilic helices as membrane interaction sites. These multivalent interactions may allow MBP to interact with two opposing membrane leaflets, contributing to the formation of a multi-layered myelin structure. The antimicrobial peptide magainin 2 interacts with the bacterial membrane, causing damage to its structure. VUVCD analysis revealed that the M2 peptides assemble in the membrane and turn into oligomers with a β-strand structure. Linear dichroism and fluorescence anisotropy suggested that the oligomers are inserted into the hydrophobic core of the membrane, disrupting the bacterial membrane. Overall, our findings demonstrate that VUVCD and its combination with theoretical and polarization experimental methods pave the way for unraveling the molecular mechanisms of biological phenomena related to protein-membrane interactions.