Multi-drug resistance (MDR) bacterial infections represent a major global health problem with increasing rate of mortality. Antimicrobial peptides (AMPs) targeted to destroy bacterial membranes have great potential as effective antimicrobial agents against MDR infections. Due to our limited understanding of the precise mechanism of action of AMPs required for improving therapeutic efficacy, no antimicrobial peptide has been approved as a drug against bacterial infection in over a decade. While alteration of the molecular organisation of lipid molecules is the major effect of AMPs resulting in loss in membrane functionality, this phenomenon is rarely measured. We have developed membrane chip technology combined with dual-polarisation interferometry (DPI) to simultaneously measure the mass bound to and the structural ordering (birefringence) of the membrane. The interaction characteristics of melittin, magainin and its analogue, HPA3, aurein 1.2, maculatin1.1 with supported planar DMPC, DMPC/DMPG (4:1), POPC, and POPC/POPG (4:1) bilayers show various degrees of disordering at different levels of bound peptide. These data allow us to determine the P/L ratio for critical threshold events corresponding to distinctive membrane structural changes related to surface binding, insertion and disruption. Further analysis of the real-time binding profiles using our recently-developed “combined mass and structural multi-state kinetics program”, reveal a three-state binding process with bilayer expansion on a POPC bilayer while at least a two-state binding process for DMPC bilayers. Overall, studying the perturbation mechanisms induced by peptides over a range of mass-density loadings provides further understanding of the mechanisms of membrane-active peptides. We also show for the first time multi-state kinetic models for simultaneous changes in bilayer structure and membrane-bound peptide which can be used to map the route of membrane destabilisation required for the rational design of membrane-destructive antimicrobial agents.