Here we developed a novel method involving the use of continuous flow for the selective alkynylation of cysteine-containing peptides and 1-thioglycoside residues. This method was characterised by mild conditions (room temperature, metal-free, oxidant-free conditions), greatly enhancing its usefulness for modifying amino acids and peptides. The newly developed S-alkynylation reagents could apparently reduce the cost and difficulty of the synthesis, thus increasing the sustainability and economy of the process. Moreover, good to excellent yields were obtained for a selection of alkynylated cysteine-containing peptides (35 examples) and 1-thioglycoside residues (15 examples) within short residence times (3–4 minutes).