Transforming Growth Factor-Beta 1 (TGF-β1) plays a pivotal role in the differentiation of fibroblasts into myofibroblasts, which is a critical process in tissue repair, fibrosis, and wound healing. Upon exposure to TGF-β1, fibroblasts acquire a contractile phenotype and secrete collagen and extracellular matrix components. Numerous studies have identified hydrogen peroxide (HO) as a key downstream effector of TGF-β1 in this pathway. HO functions as a signalling molecule, regulating various cellular processes mostly through post-translational redox modifications of cysteine thiol groups of specific proteins. In this study, we used primary human skin fibroblast cultures to investigate the oxidative mechanisms triggered by TGF-β1. We analyzed the expression of redox-related genes, evaluated the effects of the genetic and pharmacological inhibition of HO-producing enzymes, and employed an unbiased redox proteomics approach (OxICAT) to identify proteins undergoing reversible cysteine oxidation. Our findings revealed that TGF-β1 treatment upregulated the expression of oxidant-generating genes while downregulating antioxidant genes. Low concentrations of diphenyleneiodonium mitigated myofibroblast differentiation and mitochondrial oxygen consumption, suggesting the involvement of a flavoenzyme in this process. Furthermore, we identified the increased oxidation of highly conserved cysteine residues in key proteins such as the epidermal growth factor receptor, filamin A, fibulin-2, and endosialin during the differentiation process. Collectively, this study provides insights into the sources of HO in fibroblasts and highlights the novel redox mechanisms underpinning fibroblast-to-myofibroblast differentiation.