-The aim of this study was to understand development of resistance to alamethicin (a model barrel stave pore forming antimicrobial peptide) by investigating changes in phospholipid profile, fatty acid side chain analysis and extent of alamethicin insertion in biomimetic membrane prepared form wild type strains and five folds alamethicin resistant variants of Staphylococcus aureus NCDC 110, Enterococcus faecalis NCDC 114 and Bacillus cereus NCDC 66. The wild type strains NCDC 110, 114, 66, were sensitive to alamethicin with IC 50 5.5, 3.25 and 2.0 μg/ml respectively. Wild type strains were cultured in the presence of alamethicin to select resistant variants with IC 50 29.0, 17.0 and 9.5 μg/ml respectively. The phospholipid profile analysis revealed increase in amino-group containing phospholipids to amino-group lacking phospholipids ratio between wild-type and resistant variant in S. aureus and B. cereus but decreased in E. faecalis. Predominant fatty acids in all strains were composed of even number of carbons. Linoleic acid was detected only in resistant strain of B. cereus. As indicated by saturated-to-unsaturated fatty acids ratio, the membrane from S. aureus and E. faecalis became more rigid, whereas, in B. cereus it became more fluid. Using a colorimetric in vitro assay, a decrease in alamethicin insertion in the biomimetic membrane could be observed upon acquisition of resistance. The membranes of five-fold alamethicin-resistant S. aureus, E. faecalis and B. cereus revealed changes in membrane fluidity and surface charge upon acquisition of resistance to alamethicin.