A sensitive, specific and rapid HPLC-MS/MS method has been developed and validated for the simultaneous determination of cytarabine and valcytarabine (valyl prodrug of cytarabine) in rat plasma in the present study. The analytes were separated on a C18 column (50 mm x 2.1 mm, 1.7 microm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for detection. Cation exchange solid-phase extraction cartridge was employed to extract the analytes from rat plasma, with high recovery of cytarabine (>85%). The method was linear over the concentration ranges of 10-20,000 ng/mL for cytarabine and 25-1000 ng/mL for valcytarabine. The lower limit of quantitation (LLOQ) of cytarabine and valcytarabine was 10 and 25 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative error (RE) were all within 15%. Finally, the method was successfully applied to support the prodrug pharmacokinetic study after valcytarabine and cytarabine were orally administrated to the Sprague-Dawley rat, respectively.