We have developed fluorescence polarization (FP) assays of human melanocortin 4 receptor (MC4R) in 384-well microtiter plates using TAMRA-NDP-MSH as a tracer. The rank order of potency of agonists and antagonists agrees well relative to the published assays: SHU9119>MTII>NDP alphaMSH>alphaMSH. We have screened libraries of Korean plant extracts and frog peptide analogues in search of MC4R ligands using FP assays and cell-based CRE luciferase reporter assays. We report that FLGFLFKVASK, FLGWLFKVASK, FLGALFKWASK, and FLGWLFKWASK are the peptide analogues, which bind to human MC4R receptor with good affinity in vitro. FLGWLFKVASK and FLGWLFKWASK stimulated CRE-driven reporter gene via MC4R. In luciferase reporter assays, they possess the pharmacological and functional profiles of full agonists. We demonstrate the interaction of MC4R with 11-residue antimicrobial peptides derived from the Korean frog, Rana rugosa. The results suggest that MC4R interacts promiscuously with bioactive analogues of antimicrobial peptide, gaegurin-5.