Purine analogs like aracytine (AraC) are a mainstay for treating acute myeloid leukemia (AML). There are marked differences in drug dosing and scheduling depending on the protocols when treating AML patients with AraC. Large inter-patient pharmacokinetics variability has been reported, and genetic polymorphisms affecting cytidine deaminase (CDA), the liver enzyme responsible for the conversion of Ara-C to inactive uracil arabinoside (AraU) could be a culprit for either life-threatening toxicities or poor efficacy related to substantial changes in plasma exposure levels among patients. The quantitative determination of Ara-C in plasma is challenging due the required sensitivity because of the short half-life of this drug (i.e., <10 min) and the metabolic instability in biological matrix upon sampling possibly resulting in erratic values. We developed and validated a liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) for the simultaneous determination of Ara-C and Ara-U metabolite in human plasma. After simple and rapid precipitation, analytes were successfully separated and quantitated over a 1-500 ng/ml range for Ara-C and 250-7500 ng/ml range for AraU. The performance and reliability of this method was tested as part of an investigational study in AML patients treated with low dose cytarabine and confirmed marked differences in drug exposure levels and metabolic ratio, depending on the CDA status of the patients. Overall, this new method meets the requirements of current bioanalytical guidelines and could be used to monitor drug levels in AML patients with respect to their CDA phenotypes.