Abstract: Methotrexate (MTX) is a therapeutic drug that is widely used in clinic for treatment of a variety of cancers. However, MTX has limited selectivity and serious cytotoxicity, at the same time, there are problems such as narrow therapeutic window and large individual variability in metabolism and excretion of MTX. Therefore, blood concentration monitoring is required for MTX administration in clinical practice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has the advantages of strong specificity, good separation characteristics, high sensitivity and fast detection speed, which has become the internationally recognized “gold standard” for blood concentration monitoring of drugs. In this study, an isotope dilution mass spectrometry method was developed for accurate measurement of MTX in serum by the house-made QLIT-6610MD quadrupole-linear ion trap liquid chromatography-mass spectrometer. Unlike the traditional triple quadrupole QqQ tandem mass spectrometer, the quadrupole-linear ion trap (Q-LIT) mass spectrometer adopts the quadrupole-linear ion trap direct axial coupling design, i.e., a single linear ion trap is used to replace the QqQ tandem mass spectrometer’s back-end fragmentation cell and the quadrupole, and no other components are installed between the quadrupole and linear ion trap, which greatly streamlines the quality and efficiency of the mass spectrometer while ensuring the original performance of the instrument. With this unique configuration, the QLIT-6610MD instrument uses a simultaneous fragmentation and accumulation technique, which remarkedly reduces matrix-induced interferences and effectively reduces space charge effects. Serum samples were pretreated with methanol to precipitate proteins and the supernatants were measured by QLIT-6610MD spectrometer. The linear correlation coefficient (R²＞0.999 0) is obtained for MTX in the range of 10-5 000 μg/L. The limit of quantification is 10 μg/L. The precision of the quality control is better than 6.94%, and the recoveries of spiked serum samples are 86.39%-97.84% with a coefficient of variation less than 8.04%. We used the QLIT-6610MD and AB QTRAP 6500+ liquid chromatography-mass spectrometers to analyze 70 clinical serum samples for comparison of the performance of the house-made instrument by means of Pearson’s coefficient and BLand-ALtman method. The results showed that the QLIT-6610MD mass spectrometry achieves a similar level of performance to AB ATRAP 6500+ instrument, and provides a new domestic instrument option for clinical mass spectrometry. In addition, this study explores the possibility applying the multi-stage fragmentation technique (MSⁿ) to determine MTX in serum, gaining fundamental data for future studies.