<jats:title>Abstract</jats:title><jats:p>Oxidized nucleosides are biochemical markers for tumors, aging, and neurodegenerative diseases. However, during the last decade, the analytical methods for nucleosides by gas chromatography/mass spectrometry (GC/MS) and high‐performance liquid chromatography (HPLC) with single‐parameter detectors like electron‐capture detection (ECD) have not been sufficiently rapid or reliable to detect nucleosides in urine and to analyze clinical samples. It has been reported (Dudley <jats:italic>et al., Rapid Commun Mass Spectrom</jats:italic>. 2000; 14: 1200) that liquid chromatography with electrospray mass spectrometry (LC/ESI‐MS) is more specific and sensitive for analysis of nucleosides than HPLC with conventional detectors; however, this method required complex extraction steps. In the present work a direct LC/ESI‐MS method for nucleosides without extraction of urine samples has been developed. Analysis of nucleosides using positive‐ion mode with selected reaction monitoring effectively eliminated potential interferences from endogenous constituents of the urine. This highly selective and sensitive method made it possible to analyze urinary nucleosides with a lower limit of quantitation of 0.2 nmol/mL. The method has been validated, with both excellent linearity and reproducibility, in the calibration range from 0.2–400 nmol/mL. The correlation coefficients of the calibration curves were higher than 0.987. The coefficients of variation were in the range 0.03–14.92% (inter‐day) and 0.54–14.39% (intra‐day), respectively. Copyright © 2004 John Wiley & Sons, Ltd.