A key step in managing non-alcoholic fatty liver disease (NAFLD) is to differentiate nonalcoholic steatohepatitis (NASH) from simple steatosis (SS). Serum samples were collected from three groups: NASH patients (N=21), SS patients (N=38) and healthy controls (N=31). High performance liquid chromatography-mass spectrometry (HPLC-MS) was used to analyse the metabolic profile of the serum samples. The acquired data were processed by multivariate principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) to identify novel metabolites. The potential biomarkers were quantitatively determined and their diagnostic power was further validated. A total of 56 metabolites were capable of distinguishing NASH from SS samples based on the OPLS-DA model. Pyroglutamate was found to be the most promising factor in distinguishing the NASH from SS groups. With an optimal cut-off value of 4.82mmol/L, the sensitivity and specificity of the diagnosis of NASH were 72% and 85%, respectively. The area under the receiver operating characteristic (AUROC) of the pyroglutamate levels of NASH versus SS patients was more than those of tumor necrosis factor-α, adiponectin and interleukin-8. These data suggest that pyroglutamate may be a new and useful biomarker for the diagnosis of NASH.