Abstract: A urinary metabonomics method based on ultra-performance liquid chromatography-electrospray ionization quadruple time-of-flight/mass spectrometry (UPLC-ESI-QTOF/MS) was developed to study the effects of Selaginella tamariscina on hyperuricemic rats. Principal components analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) were applied to analyze the metabolites in healthy control group (HCG), model group (MG) and Selaginella tamariscina treated group (STG). The results show that significant differences in urinary metabolic profiles are observed from HCG, MG and STG by using PCA and OPLS-DA. And nine potential biomarkers including uric acid, allantoin, xanthurenic acid, kynurenic acid, indoxyl sulfate, hippuric acid, creatinine, fumaric acid and citric acid are found. Pathways of purine metabolism, tryptophan metabolism, tricarboxylic acid cycle, arginine and proline metabolism and phenylalanine metabolism are in response to the therapeutic effects of Selaginella tamariscina. Besides, the serum biochemical analysis demonstrated that Selaginella tamariscina can not only reduce the amount of uric acid but also protect the kidney of hyperuricemic rat.