This study supports the idea that BO can be initiated from a single lead peptide binder with an IC50 in the µM range and obtain peptide sequences that reach the optimal IC50s for the target in less than 5 DMT cycles. The study also shows that BO is capable of systematically finding peptide sequences with lower IC50s compared to the previous DMT cycle. Our BO method was further benchmarked by integrating predictions from different structure-based methods (Rosetta Flex ∆∆G and FlexPepDock, and FoldX), showing better performance than a sequence-based strategy (homolog scanning), but only in the situation when peptides of various lengths are considered. Luckily, the availability of a protein-peptide complex is not critical as the homolog scanning strategy is shown to be competitive against the structure-based methods when considering only fixed peptide length.