Thioamides are sterically almost identical to their oxoamide counterparts, but they are weaker hydrogen bond acceptors. Therefore, thioamide amino acids are excellent candidates for perturbing the energetics of backbone backbone H-bonds in proteins and hence should be useful in elucidating protein folding mechanisms in a site-specific manner. Herein, we validate this approach by applying it to probe the dynamic role of interstrand H-bond formation in the folding kinetics of a well-studied beta-hairpin, tryptophan zipper. Our results show that reducing the strength of the peptide's backbone backbone H-bonds, except the one directly next to the beta-turn, does not change the folding rate, suggesting that most native interstrand H-bonds in beta-hairpins are formed only after the folding transition state.