Cyclic peptides are a rich source of biologically active compounds and are produced in nature by plants, bacteria, fungi, and lower sea animals. A high-throughput methodology has been developed for the combinatorial synthesis, screening, and identification of cyclic peptide natural product analogues with improved biological activities or useful new activities. The methodology was applied to generate a library of 1716 tyrocidine A analogues, which were screened for antibacterial activity in 96-well plates. The identity of the active peptides was determined by partial Edman degradation/mass spectrometry. This has resulted in the discovery of a series of tyrocidine analogues that have significantly improved therapeutic indices compared to the natural product. The availability of tyrocidine analogues with varying antibacterial activities has provided important insights into the structure-function relationship of tyrocidine A, which should help reveal its mechanism of action.