The antibiotic activity of antimicrobial peptides is generally derived via some type of disruption of the cell membrane(s). The most common models used to mimic the properties of bacterial membranes consist of mixtures of various zwitterionic and anionic phospholipids. This approach works reasonably well for Gram-positive bacteria. However, since the membranes of Gram-negative bacteria contain lipopolysaccharides, as well as zwitterionic and anionic phospholipids, a more complex model is required to simulate the outer membrane of Gram-negative bacteria. Herein we present a protocol for the preparation of models of the outer membranes of the Gram-negative bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa. This protocol can be used to prepare models of other Gram-negative bacteria provided the strain-specific lipopolysaccharides are available.