An analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization-triple quadrupole-tandem mass spectrometry. Patient plasma samples spiked with the internal standard methotrexate were measured by multiple reaction monitoring. The detection limit was 0.4 fmol/μL, lower limit of quantification was 0.9 fmol/μL, and upper limit of quantification was 60 fmol/μL, respectively. Overall observed pemetrexed concentrations in patient samples ranged between 8.7 (1.4) and 142.7 (20.3) pmol/μL (SD). The newly developed mass spectrometric assay is applicable for (routine) therapeutic drug monitoring of pemetrexed concentrations in plasma from non-small cell lung cancer patients.