Yakuamides A (1) and B (2) possess four alpha,beta-dehydroamino acid residues in their linear tridecapeptide sequence and differ in their residue-3 (Gly for 1 and Ala for 2). The highly unsaturated peptide structure, characteristic cytotoxicity profile, and extreme scarcity from natural sources motivated us to launch synthetic studies of 1 and 2. Here, we report the total synthesis of the originally proposed structure of yakuamide B (2a) by applying the route to 1a, which was previously established in our group. However, this accomplishment only proved that 2a and natural 2 were structurally different and prompted investigations directed toward determining the true structure of 2. Extensive Marfeys analyses of minute amounts of natural 2 and its degradation products presented us the possible stereoisomers, all of which were synthetically prepared for chromatographic comparison with the authentic fragments of 2. Based on this detective work, we proposed a corrected structure for yakuamide B (2c), in which the orders of residues-7 and -8 and residues-11 and -12 are reversed. Finally, the total synthesis of 2c led to confirmation of its structural identity. Moreover, the revised structure of yakuamide A (1c) was constructed by switching Ala-3 to Gly-3 and was found to be chromatographically matched with the re-isolated natural 1. The present work demonstrated the high reliability and sensitivity of the MS- and LC-based structural analyses and the indispensable role of chemical synthesis in structural elucidation of scarce natural products.