Modified nucleosides excreted in urine have been studied as possible bio-markers for malignant tumors. Reversed-phase high performance liquid chromatography (RP-HPLC) is one of the most popular analytical tools. However, its analysis time is longer than desired. The analytical method for urinary nucleosides has been developed using a monolithic column and a linear gradient elution of 25 mmol/L KH2PO4 solution (pH 4.55) and 60% methanol in water with UV detection at 260 nm. Twelve urinary nucleosides were completely separated. The monolithic column presented similar analytical characteristics to reversed-phase column. The time of the analysis is only 23 min, in general much shorter than the methods using a packed RP-HPLC. The method developed is sensitive, reliable and suitable for clinical applications.