A comprehensive understanding of peptide behavior required a combination of experimental techniques (e.g., NMR) and computational methods (e.g., MD simulations, MHP mapping). Their application to the most potent peptides and corresponding analogs provided insights into structural differences that influence their biological profiles, including peptide conformation, flexibility, surface amphiphilicity, and oligomerization tendencies. In summary, it can be assumed with some confidence that peptides with limited flexibility and side-chain packing that creates a smaller, more fragmented surface hydrophobic pattern are less likely to insert into eukaryotic membranes, thereby exhibiting reduced toxicity. The key finding of this study is that the hemolytic activity of cyclic amphiphilic peptides (at least within the current extensive dataset) can be reliably predicted by considering the nuanced interplay of structural and hydrophobic parameters derived from computer simulations guided by NMR data.