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Effective antimicrobial peptides (AMPs) distinguish between the host and microbial cells, show selective antimicrobial activity and exhibit a fast killing mechanism. Although understanding the structure–function characteristics of AMPs is important, the impact of the peptides on the architecture of membranes with different lipid compositions is also critical in understanding the molecular mechanism and specificity of membrane destabilisation. Real-time quantitative analysis of the structural properties of membrane organisation has allowed the membrane destabilisation process to be resolved into multiple steps and provides comprehensive information to determine the molecular mechanism of antimicrobial peptides.