For 70 years, α-amanitin, the most cytotoxic peptide in its class, has been without a synthetic rival; through synthesis, we address the structure-activity relationships to inform the design of new amatoxins and disclose analogues that are more cytotoxic than the natural product when evaluated on CHO, HEK293, and HeLa cells, whereas on liver-derived HepG2 cells, the same toxins show diminished cytotoxicity.