Abstract: It is widely known that reactive oxygen species (ROS), such as hydrogen peroxide, play important roles in cellular signaling and initiation of oxidative stress responses via thiol modifications. Identification of the targets of these modifications will provide a better understanding of the relationship between ROS and human diseases, such as cancer and atherosclerosis. Sulfenic acid is the principle product of a reaction between hydrogen peroxide and a reactive protein cysteine. This reversible post-translational modification plays an important role in enzyme active sites, signaling transduction via disulfide bond formation, as well as an intermediate to overoxidation products during oxidative stress. By re-engineering the C-terminal cysteine rich domain (cCRD) of the Yap1 transcription factor, we were able to create a genetically encoded probe for the general detection and identification of proteins that form sulfenic acid in vivo. The Yap1-cCRD probe has been used previously in the identification of proteins that form sulfenic acid in Escherichia coli. Here we demonstrate the successful use of the Yap1-cCRD probe in the identification of proteins that form sulfenic acid in response to hydrogen peroxide in Saccharomyces cerevisiae.