Background and aims: A selective and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been established for determination of methotrexate in human plasma. Methods: Methotrexate was extracted from plasma with acetonitrile. The mobile phase consisted of acetonitrile-water-formic acid 74: 25: 1(v/v). Twenty µl of sample was chromatographically analyzed using a repacked ZORBAX-XDB-ODS C18 column (2.1mm×30mm, 3.5 micron). The mode of mass spectrometry was selected-ion monitoring (SIM). Results: Standard curve was linear (r= 0.998) over the concentration range of 0.1–100.0 ng/ml and showed a suitable accuracy and precision. The limit of detection (LOD) was 0.05ng/ml. Conclusion: The method is quick, easy, very steady and precise for the partition, assignment and evaluation of methotrexate in human plasma
