OBJECTIVE The precise identification of many diseases of the gastrointestinal tract requires the histological analysis of multiple biopsies of the lining mucosae, thus preventing an immediate diagnosis and the safe screening of the entire organ. To address these limitations, we developed a novel spectroscopic procedure for a real-time, non-invasive optical analysis of mucosae. METHODS We have used a fibre-optic probe that monitors light propagation through small tissue volumes to evaluate the antral and fundic mucosa of 51 patients that underwent gastroscopy for symptoms of dyspepsia. Several optical coefficients were computed from the recorded light reflectance, and confronted to the diagnosis made by an expert gastroscopist at the time of the clinical examination. Both evaluations were then validated by comparison with the histological diagnosis of a pathologist who screened biopsies taken at the sites of the optical measurements. RESULTS We report that the optical procedure discriminated normal and pathological gastric mucosae with a higher sensitivity and specificity than endoscopic diagnosis. We also show that the changes in light-scattering coefficient, which permitted the optical diagnosis of gastritis alterations, were indirectly correlated with the extent of inflammatory infiltration of the mucosa and detected mucosal alterations mild enough to escape endoscopic detection. CONCLUSIONS The results show that, in a normal clinical setting, the optical in vivo analysis provided by our system detects alterations typical of gastritis, and allow for their graded scoring with a specificity and sensitivity that compare well with those of standard histology, while avoiding the invasiveness of the latter procedure. The method is adaptable to the screening of other types of lesions and mucosae and, hence, should prove useful in improving available diagnostic approaches.