In vitro selection methods represent a powerful approach toward identifying high-affinity peptide ligands from highly diverse peptide libraries against a desired target. We herein describe a method for the display and selection of cyclic thioether peptide libraries. Reprogramming the initiation event from fMet to an N-chloroacetyl-amino acid by utilizing flexizyme to rapidly and efficiently prepare the aa-tRNA can be effectively used to initiate translation, upon which the thiol group of an inserted cysteine at the C terminus of the designed library spontaneously reacts to yield a nonreducible cyclic thioether peptide readily compatible with any in vitro display methods. Thus, cyclic peptides already in a nonreducible stable form can be selected directly against the target of interest.