An ultra high performance liquid chromatography-tandem mass spectrometric(UPLC-MS/MS) method was developed for the simultaneous determination of twelve normal and modified nucleosides in mRNA of cancer cells.Total RNA extraction,mRNA purification and mRNA hydrolyzation were performed using cultured cancer cell samples from commercial kits,with 8-bromoguanosine(8Br-G) as internal standard.Twelve normal and modified nucleosides were separated on an Agilent RRHD SB-C18(2.1 mm×50 mm,1.8 μm) column with 0.1% formic acid methanol solution and 0.1% formic acid solution as mobile phases,and determined by UPLC-MS/MS with positive electrospray ionization(ESI+) under multiple reaction monitoring(MRM) mode.The method could meet the requirements for method validation.There were good linear relationships for twelve normal and modified nucleosides in mRNA of cancer cells in the certain concentration ranges with their correlation coefficients(r) larger than 0.99.The limits of quantitation and limits of detection were in the ranges of 0.17-3.13 ng/mL and 0.08-1.56 ng/mL,respectively.The method was then applied to human nasopharyngeal carcinoma 5-8F cell line,human cervical cancer Hela cell line and human embryonic kidney epithelial 293T cell line,with successful determination of various modified nucleosides.The method is capable of simultaneous quantitation for twelve normal and modified nucleosides in mRNA with short analysis time(6 min per sample),low number of cells(2×106 per sample) and high sensitivity,which could provide a powerful tool for cancer research and clinical diagnosis.