Determination of nucleosides and their metabolic compounds is important for physiological and pharmacological studies. Herein, a rapid ultra-performance liquid chromatography (UPLC) method was developed for the simultaneous determination of 14 nucleosides and nucleobases, namely adenine, adenosine, cytosine, cytidine, uracil, uridine, guanine, guanosine, hypoxanthin, inosine, thymine, thymidine, 2′-deoxyuridine and cordycepin. The separation was performed on Waters Acquity UPLC system with Acquity UPLC BEH C18 column and gradient elution of 0.5 mM acetic acid and acetonitrile in 5 min. The correlation coefficients of 14 analytes were high (R2 > 0.9995) within the test ranges. The LOD and LOQ were lower to 11.9 and 47.0 ng/ml with 1 μl of injection volume, respectively. The overall R.S.D. for intra- and inter-day of 14 analytes were less than 1.8%. The developed method was applied for the analysis of nucleosides and nucleobases in cultured Cordyceps, which also could be used for the fast determination of the analytes in pharmaceutical products and biological fluids. © 2007 Elsevier B.V. All rights reserved.