We report a streamlined synthesis of (2S,3R,4R)-4,5-dihydroxy isoleucine (DHIle), an amino acid found in α-amanitin, which appears to be critical for toxicity. This synthetic route is transition metal-free and enables the production of significant quantities of DHIle with suitable protection for use in peptide synthesis. Its incorporation into a cytotoxic amatoxin analog is reported.