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 action mechanisms of AMPs required for effective therapeutics, 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 and correlate the mass of peptides bound to and the changes in 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 correlation profiles with distinctive degrees of disordering at different levels of bound peptide. These distinctive membrane disordering data allow us to determine sequential P/L threshold events corresponding to surface binding, insertion and disruption. Further analysis of the real-time binding profiles using our recently-developed “mass and structure-correlated multi-state kinetics program”, reveal a three-state binding process with bilayer expansion on a fluid-state POPC bilayer while at least a two-state binding process on gel-state DMPC bilayers. Overall, correlating the membrane disordering over a range of mass-density loadings provides better understanding of the membrane perturbation mechanisms of membrane-active peptides. We have used, for the first time, the multi-state kinetic models to quantitate simultaneously changes in bilayer structure and membrane-bound peptide mass which can be used to map the route of membrane destabilisation required for the rational design of membrane-destructive antimicrobial agents.