Determination of the liver histological lesions with noninvasive tests is an important part of the diagnostic work-up of patients with non-alcoholic fatty liver disease (NAFLD). We aimed to determine the predictive value of noninvasive biochemical markers, serum prolidase enzyme activity (SPEA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and AST/ALT ratio for the liver histological lesions. Fifty-four liver biopsy-proven patients with NAFLD and 37 healthy controls were enrolled to the study. The diagnostic accuracies of biochemical markers were evaluated by receiver operating characteristic (ROC) curves and multiple linear regression analysis to predict the degree of fatty infiltration, lobular inflammation, NAFLD activity score, and stage of fibrosis. The SPEA of patients with steatohepatitis is significantly increased compared with the patients with simple steatosis and controls (1,338 [1,138-1,624] U/l; 974 [768-1,160] U/l; 972 [862-1,122] U/l, shown as median [25th-75th interquartile range], respectively, P < 0.0001). SPEA was positively correlated with the grade of liver fatty infiltration, lobular inflammation and NAFLD activity score, and stage of fibrosis, (r = 0.377, P < 0.005; r = 0.443, P < 0.001; r = 0.457, P < 0.001; r = 0.321, P < 0.018, respectively). SPEA was the best predictor for distinguishing steatohepatitis from simple steatosis according to the ROC analysis (area under the curve [AUC]: 0.85). Multivariate analysis revealed that the most useful single test for predicting lobular inflammation, NAFLD activity score, and fibrosis was SPEA, and for predicting the fatty infiltration, it was ALT (P < 0.00001, P < 0.001, P < 0.0001, P < 0.0001, respectively). This study demonstrated that SPEA can accurately predict the degree and stage of all histological lesions in NAFLD. It could be helpful for distinguishing steatohepatitis from simple steatosis and reducing the need for liver biopsy in the majority of patients with NAFLD.