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Various diseases and immune-related issues have been associated with the gastrointestinal system’s health. Gastrointestinal permeability - a measure of transport across the GI tract’s cell lining from the lumen - is a functional parameter that is affected and is a viable factor in GI health assessment. Current diagnosis of GI-related disease involves the use of invasive exploratory surgery to minimally invasive colonoscopy. Magnetic particle imaging (MPI) is a non-radioactive and highly sensitive tracer imaging modality. The nanoparticle tracers used for MPI are FDA approved and can readily be translated into the clinic. In this research, we provide the first proof-of-concept using MPI to evaluate GI permeability in an in vitro model of the epithelial barrier lining of the GI tract.