Combination of cytostatic agents is a basic principle in the treatment of cancer. For the treatment of acute myeloid leukemia (AML), purine analogs, like clofarabine and cytarabine act synergistically. Little is known, however, on their interaction in vivo. We developed a method for the simultaneous determination of clofarabine and cytarabine in human plasma. The substances were extracted from plasma samples by protein precipitation with acetonitrile. Cladribine was the internal standard (IS). The analytes were separated on Synergi HydroRP column (150 mm × 2.0 mm, 4 μm) and a triple-quadrupole mass spectrometry with an electrospray ionisation (ESI) source was applied for detection. The mobile phase consisted of acetonitrile, ammonium acetate 2 mM and 0.5% formic acid in a gradient mode at a flow rate of 0.5 ml/min. The injection volume was 10 μl and the total run time was 6.0 min. Retention times were 2.46 min for clofarabine, 0.97 min for cytarabine and 2.43 min for the IS. Calibration ranges were 8-1000 ng/ml for clofarabine and 20-2500 ng/ml for cytarabine. The intra-day and inter-day precision was less than 15% and the relative standard deviation was all within ±15%. This new method allows a rapid and simple determination of both clofarabine and cytarabine in human plasma. It was applied to a pharmacokinetic investigation within a hematological trial in adult patients with AML.