An ion-pair reversed phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the quantification of a phosphorothioate oligonucleotide (PS-OGN) PF-ODN and its metabolites 5'N-1/3'N-1, 5'N-2 and 5'N-3 in rat plasma. Plasma samples were prepared with an initial phenol/dichloromethane liquid-liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a Phenomenex Gemini C(18) column using hexafluoro-2-propanol/triethylamine buffer and methanol as the mobile phase at a flow rate of 0.5mL/min. Except for 5'N-1 and 3'N-1, which were coeluted and could not be differentiated by mass spectrometer, the other analytes were separated chromatographically and mass spectrometrically. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) achieved was 4.0ng/mL for PF-ODN and its four metabolites with acceptable precision and accuracy. Inter-day and intra-day RSD for three quality control (QC) levels across validation runs were less than 12.0% and the accuracy ranged from -9.6% to 6.0% for the analytes. This validated LC-MS/MS method was applied to a preliminary pharmacokinetic study of PF-ODN in rats.