BACKGROUND: Liver biopsy is considered the gold standard for assessing histologic lesions of non-alcoholic fatty liver disease (NAFLD). The aim was to develop and validate a new biomarker of non alcoholic steato hepatitis (NASH) the NashTest (NT) in patients with NAFLD. METHODS: 160 patients with NAFLD were prospectively included in a training group, 97 were included in a multicenter validation group and 383 controls. Histological diagnoses used Kleiner et al's scoring system, with 3 classes for NASH: "Not NASH", "Borderline", "NASH"). The area under the ROC curves (AUROC), sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV, NPV) were assessed. RESULTS: NT was developed using patented algorithms combining 13 parameters: age, sex, height, weight, and serum levels of triglycerides, cholesterol, alpha2macroglobulin, apolipoprotein A1, haptoglobin, gamma-glutamyl-transpeptidase, transaminases ALT, AST, and total bilirubin. AUROCs of NT for the diagnosis of NASH in the training and validation groups were, respectively, 0.79 (95%C I 0.69-0.86) and 0.79 (95% CI 0.67-0.87; P = 0.94); for the diagnosis of borderline NASH they were: 0.69 (95% CI 0.60-0.77) and 0.69 (95% CI 0.57-0.78; P = 0.98) and for the diagnosis of no NASH, 0.77 (95% CI 0.68-0.84) and 0.83 (95% CI 0.67-0.90; P = 0.34). When the two groups were pooled together the NashTest Sp for NASH = 94% (PPV = 66%), and Se = 33% (NPV = 81%); for borderline NASH or NASH Sp = 50% (PPV = 74%) and Se = 88% (NPV = 72%). CONCLUSION: In patients with non-alcoholic fatty liver disease, NashTest, a simple and non-invasive biomarker reliably predicts the presence or absence of NASH.