Non-Celiac Gluten Sensitivity (NCGS) is a common disorder characterized by symptoms resembling those of irritable bowel syndrome. In recent years there has been progress in the understanding of the pathogenic pathways and data suggest that NCGS has a distinct immunological profile that differs from celiac disease (CeD). This has fostered the search for a specific biomarker of NCGS. In this review we will firstly update on pioneer NCGS diagnostic tools, particularly the gluten challenge, serum IgG class antigliadin antibodies, and certain histological characteristics seen at the small intestinal biopsy. Then we will examine the most recent research on potential biomarkers of NCGS, specifically focusing on markers of damage to enterocytes, of translocation of bacteria from the gut into the bloodstream, intestinal permeability, and inflammation. So far, no specific biomarker of NCGS has been detected. The diagnosis of NCGS still relies on clinical criteria. A gluten challenge may be useful for diagnostic purposes, however a strong nocebo effect limits the efficacy of this procedure. Additional investigation is necessary to identify biomarkers for NCGS, that may be useful to investigate the epidemiology, clinical spectrum, and natural history of this common disorder.