An improved liquid chromatography-mass spectrometry method for the determination of pemetrexed in human plasma was developed and validated using a simple quadrupole LC-MS and a new SPE cartridge (Plexa Bond Elut). The analysis was achieved with a C18 analytical column using a mobile phase consisting of formic acid/acetonitrile and isocratic flow for 7 min. The linear ranges (r(2)>0.99) were found from 5 to 5000 ng/mL. The lower limit of detection was 2.5 ng/mL. Within-day and between-day precisions were less than 7.2% and inaccuracy did not exceed 2.8%. This new method is suitable to support pharmacokinetic studies and drug monitoring.