Amphiphilicity is a crucial factor in the mechanism of action of antimicrobial peptides (AMPs). If the secondary structure of AMPs is not properly maintained, their amphiphilicity can be compromised.1 This highlights the importance of post-synthetic modifications that stabilize the secondary structure, as they can enhance bioactivity and improve resistance to proteolytic degradation. One effective method to achieve this is through peptide stapling – a range of macrocyclization techniques that allow for the covalent linking of two side chains within the peptide sequence.2 While peptide stapling is often utilized to modify α-helices, it should be noted that many AMPs, such as gramicidin S (GS), exhibit a β-sheet structure. This represents an important area within peptide stapling that has yet to be thoroughly investigated.3 In our research, we developed two novel stapled analogs of GS, each with varying staple lengths that contributed to different levels of structural rigidity. We began by substituting Leu residues with Cys residues, following this substitution, we incorporated one of two stapling methods. The first method involved the introduction of a perfluoroaryl bridge via thiolate moieties, while the second method focused on forming a disulfide bridge between the Cys residues. We systematically assessed the bioactivity and structural modifications of the synthesized peptides, comparing them against unmodified GS and a linear analog known for its enhanced flexibility. This study aimed to shed light on the structure-activity relationship of β-sheet peptides, especially regarding how changes in rigidity influence their antimicrobial properties. Moreover, we conducted molecular dynamics simulations in both aqueous and membrane environments to complement our experimental results. Our findings indicate that conformational flexibility may significantly affect the activity of peptides, playing a role that is comparable to the importance of hydrophobicity.4