Purpose: A simple, rapid and reliable method to quantify methotrexate (MTX) in human plasma by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS /MS) was established and validated in two laboratories. Patients and Methods: Sample separation was achieved on a Synergi Hydro-RP column (50 mmx2.0 mm, 2.5 mu m) with a gradient elution program in 3.5 mM after a simple protein precipitation with methanol (MeOH) and acetonitrile (ACN) (1:1). About 5 mM ammonium formate aqueous solution with 0.2% formic acid and ACN were used as mobile phase with a flow rate of 0.5 mL/min at 40 degrees C. Mass spectrometry detection using AB Sciex Triple Quad 4500 mass spectrometer (4500 QQQ) and Q-trap 5500 mass spectrometer (5500 Q-trap) were both characterized by electrospray ionization (ESI) for positive ions in multiple reactionmonitoring (MRM) mode. Quantitative ion pairs were m/z 455.1 -> m/z 308.0 for MTX and m/z 248.1 -> m/z 121.0 for tinidazole (TNZ) used as internal standard (IS). Results: Linear calibration curves were generated over the range of 5-1000 ng/mL (r(2) > 0.99) on both the 4500 QQQ and 5500 Q-trap, both of the intra- and inter-batch precision were less than 7.67% and accuracy ranged from 96.33% to 108.94%. The recovery and matrix effect were 82.20-93.98% and 102.69-105.28%, respectively. Conclusion: An analytical method transfer was achieved by re-verification in two laboratories to ensure stability and reproducibility and this method has been applied for therapeutic drug monitoring (TDM) successfully in children and adults with NHL, and during routine TDM, two delayed elimination of MTX cases were observed and analyzed.