Chemical modification of biomolecules such as peptides and proteins is a fundamental technology for creating new functional molecules. Recently, methods targeting tryptophan (Trp) residues for chemical modification have gained attention. Trp appears to be an ideal target for amino acid–specific and –selective modification due to the inherent reactivity of its electron-rich indole side chain and its low natural abundance among all amino acids in eukaryotes (approximately 1%)[1]. Therefore, development of specific and selective reactions targeting Trp could offer promising strategies for biomolecular modification. To this end, several methods have been developed, including oxidation of the Trp indole ring using oxaziridines (Trp-CliC)[2] and sulfenylation with S-protected cysteine sulfoxides[3]. Recently, we discovered that 4-fluorophenyl 3-nitro-2-pyridinesulfenate (Npys-OPh(pF)) functions as a novel sulfenylation reagent for the Trp indole ring, activated by thioethers such as methionine or diethyl sulfide under acidic conditions[4]. We investigated the substrate scope of this sulfenylation using various Trp-containing bioactive peptides. Our results showed that Trp-sulfenylation proceeded efficiently, yielding the desired modified peptides. Furthermore, we expanded this sulfenylation to the late-stage chemical modification of the Trp-containing natural cyclic depsipeptide, daptomycin and successfully obtained derivatives bearing longer alkyl chains via the Npys structure, as well as further functionalization through fluorescent labeling. The thioether-mediated sulfenylation, as a robust chemical modification for Trp-containing biomolecules, could be further extended to a wide range of applications in medicinal chemistry, bioimaging, and chemical biology.