Despite the intensive study of antibiotic-induced bacterial permeabilization, its kinetics and molecular mechanism remain largely elusive. A new methodology that extends the concept of the live-dead assay in flow-cytometry to real time-resolved detection was used to overcome these limitations. The antimicrobial activity of pepR was monitored in time-resolved flow cytometry for three bacterial strains: E. coli (ATCC 25922), E. coli K-12 (CGSC Strain 4401) and E. coli JW3596-1 (CGSC Strain 11805). The latter strain has truncated lipopolysaccharides (LPS) in the outer membrane. This new methodology provided information on the efficacy of the antibiotics and sheds light on their mode of action at membrane-level. Kinetic data regarding antibiotic binding and lytic action was retrieved. Membrane-interaction and permebilization events differ significantly among strains. The truncation of LPS moieties does not hamper AMP binding but compromises membrane disruption and bacterial killing. We demonstrated the usefulness of time-resolved flow cytometry to study antimicrobial-induced permeabilization by collecting kinetic data that contribute to characterize the action of antibiotics directly on bacteria.