A sulfur-containing analog of tryptophan, thiotryptophan (3-benzothienyl-L-alanine, S-Trp), was genetically incorporated into EcKatG to replace the MYW-forming tryptophan, W105. The heme free apo-protein was purified, after heme-reconstitution and peroxide treatment, the S-Trp-bearing peptide was isolated using trypsin digestion followed by HPLC separation. Tandem mass spectrometry (MS/MS) was then performed to verify the substitution. The final results demonstrated that S-Trp was successfully incorporated and subsequently underwent S-monooxygenation, yielding O=S-Trp.