Enfuvirtide is the first approved membrane fusion inhibitor against HIV-1. Although this drug is effective against multi-drug resistant strains, the emergence of enfuvirtide-resistant strains has been reported in patients who have received an enfuvirtide-containing regimen. Based on the high affinity of synthetic HIV-1 gp41 C-terminal heptad repeat (C-HR) peptides to the counterpart trimeric N-terminal heptad repeat (N-HR) coiled-coil structure, a novel screening approach has been established to facilitate the identification of potent fusion inhibitors against wild-type and enfuvirtide-resistant HIV-1. In this process, affinity selection using histidine-tagged N-HR peptides with the sequences derived from wild-type and resistant strains efficiently captured potent inhibitory peptides from a pool of highly water-soluble C-HR peptides with α-helix-inducible motifs. A highly potent peptide was found from a single amino acid substitution observed in an enfuvirtide-resistant variant as well as peptides with unprecedented modifications at the mutated site.