Celiac disease (CD) is an intestinal autoimmune disorder developed in genetically susceptible individuals upon gluten ingestion. Gliadin is known to be the most immunogenic gluten component, which can activate the host immune response represented by NFkB activation and release of proinflammatory cytokines as IL8. However, many aspects of the involvement of gliadin in CD pathophysiology is not well understood yet. Lack of a CD animal model increases difficulty elucidating key steps in CD development, what increases the importance of in vitro experiments. Here we present a protocol for in vitro pepsin-trypsin digested gliadin (PTG) treatment for long term studies in HCT116 intestinal cell line.