The cell adhesion protein nectin-4 emerged as a valid therapeutic target for antibody- and peptide-drug conjugates in cancer. To support patient stratification for such targeted therapies, there is a clinical need for molecular imaging agents capable of quantifying nectin-4 levels noninvasively in vivo. For this purpose, we developed⁶⁴Cu- and⁶⁸Ga-labeled ligands derived from bicyclic peptide-drug conjugate BT8009. A library of peptides was prepared with a major focus on the bioisosteric replacement of the original methionine residue due to its susceptibility to oxidation. The peptides were characterized for their binding behavior to nectin-4, and radiopharmacological characterization of selected radioligands was performed using urothelial carcinoma cell lines and tumor xenograft models derived thereof. The suitability of the most promising ligand from the preclinical studies, NECT-224, for PET imaging purposes was also demonstrated in a first-in-human application using [⁶⁸Ga]Ga-NECT-224. The results suggest its further clinical development, but also that of [⁶⁴Cu]Cu-NECT-224.