Gastrointestinal disorders such as inflammatory bowel disease and irritable bowel disorder cause a substantial economic and social burden and affect a large population. Impairment of the mucosal barrier is a common underlying cause for such disorders and enhancement of gastrointestinal epithelial repair is a promising therapeutic strategy. The trefoil factor (TFF) peptides are key players in protecting, maintaining and repairing the gastrointestinal tract, but their mechanisms of action are not fully understood. The members of this peptide family share a highly conserved structural motif named trefoil-domain or P-domain that comprises 42-43 amino acids, including six cysteine residues whose spacing pattern is conserved (CX9-10C9CX4CCX10C) to form three intra-chain disulfide bonds in the configuration Cys1-5, Cys2-4 and Cys3-6. In addition to the cysteine residues inside the trefoil-domain, TFF1 and TFF3 have an extra cysteine which can form dimers. Many studies have highlighted TFFs’ therapeutical potential for gastrointestinal disorders, however, the lack of knowledge about their mechanisms of action hampers further development. To gain more insight about this peptide family, we chemically synthesised TFF1 and TFF3. The full-length peptides (59 and 60 amino acids long) were split into two fragments which were synthesised by Fmoc-solid phase peptide synthesis, successfully ligated using native chemical ligation, and subsequently folded. To investigate if the synthetic peptide retained the biological activity, migration assays using gastrointestinal cell lines were carried out. Additionally, LC-MS was performed to assist the determination of the disulfide bond connectivities and NMR to structurally characterize the synthetic peptide. The trefoil factors represent a great and innovative opportunity for novel drug design and a better understanding of this peptide family will allow to develop therapeutic leads to tackle gastrointestinal disorders.