The library of one type of cyclic peptide immobilized on a gel-type bead (OPOB) is a powerful tool for the discovery of interacting peptides. Hydrophilic, water-swellable beads are particularly well suited for bioassays with bead-bound peptides in aqueous media, particularly with targets such as whole living cells. The library is constructed by the “split and combine method.” The key issues of successful discovery are both the quality of individual peptides on beads and the uniformity of each split batch. Two types of OPOBs have been proposed. Natural and non-natural amino acids, 19 and 5 respectively, have been used as building blocks to generate the diversity of ca. 200 million hexapeptides with cysteinyl residues forming the cyclic peptides. Selected beads after screening were sequenced initially by conventional Edman degradation, although this was time-consuming and several problems are known. To resolve these, partial acid hydrolysis with 5.7 mol/L hydrogen chloride followed by LC/MS has been proposed. In addition to the cyclic form prepared via the disulfide of cysteinyl residues, a thioether linkage was also considered. For a second generation of libraries, a methionine residue was inserted for the site-specific rapid cleavage by cyanogen bromide to liberate the immobilized peptides allowing high-throughput characterization by MALDI-TOF-MS/MS without LC purification. Peptides discovered from these libraries can be built into affinity probes for target purification, as well as cell-targeting constructs for cell-specific staining or delivery of diverse cargos, which has been designated “peptide-vehicle” for therapy.