Cationic antimicrobial peptides (CAMPs) are potent therapeutics for drug-resistant bacterial infections. However, the clinical application of CAMPs is hampered by its poor proteolytic stability and hemolytic activity toward eukaryotic cells. Great efforts have been made to design and generate derivatives of CAMPs with improved pharmacological properties. Here, we report a novel stapling protocol, which tethers two ε-amino groups of the lysine residue by the N-alkylation reaction on the hydrophilic face of amphiphilic antimicrobial peptides. A series of lysine-tethered stapled CAMPs were synthesized, employing the antimicrobial peptide OH-CM6 as a model. Biological screening of the stapled CAMPs provided an analogue with strong antimicrobial activity, high proteolytic stability, and low hemolytic activity. This novel stapling approach offers an important chemical tool for developing CAMP-based antibiotics.