The first total synthesis of cyclic depsipeptide antibiotic LL-A0341β1 (LL) is described. The configuration of the β-methyltryptophan (β-MeTrp) residue was established by preparing all four stereoisomers of Fmoc-β-MeTrp which were used for the synthesis of LL via Fmoc solid phase peptide synthesis. The most active of the four peptides was the one containing (2R,3R)-β-MeTrp. The activity of LL was strongly inhibited by cardiolipin (CL), but not other common phospholipids found in bacterial cell membranes, suggesting that LL interacts with CL in the lipid membrane.