Opioid receptor screening of a synthetic conopeptide library led to the discovery of conorphin-Tx as a novel selective k-opioid agonist lead. Selective disulfide bond formation, alanine scanning, peptide truncations, backbone modifications, disulfide mimicry, and the use of unusual amino acid building blocks generated a pharmacophore model that allowed the design of highly potent and selective k-opioid receptor (KOR) agonists. Using iterative synthesis and pharmacological evaluation in parallel with stability testing resulted in potent KOR agonists with vastly improved potency and plasma stability. The peptidic KOR agonists discovered are defined by a hydrophobic benzo-prolyl moiety, a double arginine sequence, a spacer amino acid followed by a hydrophobic residue and a C-terminal vicinal disulfide moiety essential for positioning the C-terminal amide group. Our pharmacophore model was supported by computational docking studies that revealed receptor-ligand interactions similar to those of the KOR agonist dynorphin A (1-8). A selected conorphin agonist inhibited colonic nociceptors in a mouse tissue model of chronic visceral hypersensitivity, highlighting the potential of developing KOR agonists for the treatment of chronic abdominal pain. This new class of vicinal-disulfide-containing KOR agonist peptides and the elaborated pharmacophore provide new opportunities for rational opiate receptor drug development and a set of potentially peripherally-restricted probes for exploring the role and mechanism of action of k-opioid receptors.