A LC-MS/MS method was developed and validated for determination of nucleoside analog (NA) in rat plasma. The method run time was 6 min and the limit of quantification (LOQ) was estimated at 100 pg/mL. The extraction procedure consisted on plasma samples protein precipitation with an acetonitrile solution which contained the stable isotope labeled internal standard (IS). Chromatography was performed on a newly developed C(16) column (150 mm x 4.6 mm, 5 microm) in order to avoid the use ion pair salts. The samples were eluted at 0.8 mL/min with a gradient of mobile phase made of water and acetonitrile both acidified with 0.5% acetic acid and 0.025% trifluoroacetic acid (TFA). A tandem mass spectrometer was used as a detector for quantitative analysis. Intra-run and inter-run precision and accuracy within +/-15% were achieved during a 3-run validation for quality control samples at four concentration levels in rat plasma, over a concentration ranging between 0.1 and 1000 ng/mL. The data indicate that our LC-MS/MS assay is an effective method for the pharmacokinetics study of NA in rat plasma.