<p>The application of membrane-active antimicrobial peptides (AMPs) is considered to be a viable alternative to conventional antibiotics for treating infections caused by multidrug-resistant pathogenic microorganisms. In vitro and in silico biophysical approaches are indispensable for understanding the underlying molecular mechanisms of membrane-active AMPs. Lipid bilayer models are widely used to mimic and study the implication of various factors affecting these bio-active molecules, and their relationship with the physical parameters of the different membranes themselves. The quality and resemblance of these models to their target is crucial for elucidating how these AMPs work. Unfortunately, over the last few decades, no notable efforts have been made to improve or refine membrane mimetics, as it pertains to the elucidation of AMPs molecular mechanisms. In this review, we discuss the importance of improving the quality and resemblance of target membrane models, in terms of lipid composition and distribution, which ultimately directly influence physical parameters such as charge, fluidity, and thickness. In conjunction, membrane and peptide properties determine the global effect of selectivity, activity, and potency. It is therefore essential to define these interactions, and to do so, more refined lipid models are necessary. In this review, we focus on the significant advancements in promoting biomimetic membranes that closely resemble native ones, for which thorough biophysical characterization is key. This includes utilizing more complex lipid compositions that mimic various cell types. Additionally, we discuss important considerations to be taken into account when working with more complex systems.</p>