In this study, we designed and synthesized a series of novel AMPs based on peptide 1, which was developed in our previous study, by replacing Lys residues with other cationic amino acids, Orn, Dab, Arg, and His. The preferred secondary structure of synthesized peptides 1–5 was investigated. As shown in Figure 2, peptide 1–5 adopted a stabilized helical structure through the introduction of two Aib residues into the sequence. It has been demonstrated that replacement of Lys residues with other cationic amino acids has no significant effect on their helical structure. The antimicrobial and hemolytic activities of the synthesized peptides were also evaluated. It has been revealed that peptides 2, 3, and 4, which have Orn, Dab, and Arg residues, respectively, showed the same extent of antimicrobial activity against Gram-positive and Gram-negative bacteria as peptide 1. In contrast, peptide 5, which has His residues, showed no or weak antimicrobial activity. Because AMPs target bacterial membranes, they also affect human cell membranes. Therefore, we evaluated the hemolytic activity of the synthesized peptides against human red blood cells. Generally, an increase in the hydrophobicity of amphipathic peptides tends to enhance their hemolytic activity [25]. Based on this knowledge, it was expected that peptides 2 and 3 would exhibit lower hemolytic activity due to their shorter alkyl side chains. Surprisingly, it was observed that the peptides 2 and 3 with shorter alkyl chain exhibited higher hemolytic activity than peptide 1. It remains unclear why peptide 3 exhibited stronger hemolytic activity than peptide 1, which may affect its affinity for the human membrane. Finally, the chemical stability of peptides 1 and 2 was investigated to confirm the utility of substituting Lys residues with Orn residues. Although peptide-based drugs show excellent efficacy, one of the challenges is that they are easily degraded by digestive enzymes [24]. Orn is a non-proteinogenic amino acid that is difficult to recognize for digestive enzymes and increases chemical stability of Orn-containing peptides. As shown in Figure 3, peptide 2 exhibited a longer t1/2 than peptide 1, indicating that the replacement of Lys residues with Orn residues increased the chemical stability without any significant effects on their secondary structure, antimicrobial activity, and hemolytic activity. These results suggest that replacement of Lys residues with Orn residues could be a promising strategy for the development of AMPs.