Methotrexate (MTX) is a dihydrofolate reductase inhibitor that is used for the treatment of tumors and autoimmune diseases. Several automated binding assays are used in clinical practice and numerous chromatographic methods have been developed toward higher specificity and sensitivity. In the present study, phenyl cartridges were used for the solid-phase extraction (SPE) of MTX from human serum samples; subsequently, extracts were analyzed by reversed-phase high-performance liquid chromatography. Isocratic separation was implemented on a Kromasil-C18 column with a mobile phase consisting of 50 mM sodium acetate buffer (pH 3.6)-acetonitrile (89:11, v/v) and ultraviolet detection at 307 nm. MTX eluted in less than 12 min with no interference from impurities or 24 examined drugs. Detector response was linear in the range of 0.025-5.00 µΜ (coefficient of correlation > 0.99). Recovery from the serum was 93.1-98.2% and bias was < 8.3%. Intra-day and inter-day precision were <7.8 and 12.6%, respectively (n = 6). The limit of quantitation was 0.01 µM and the limit of detection was 0.003 µΜ. The method was validated by using serum samples from osteosarcoma patients treated with high-dose MTX (8-12 g/m(2)). In conclusion, the combined use of a phenyl-functionalized sorbent for SPE and a Kromasil-C18 column, and specific detection at 307 nm, assured a selective, fast, robust and cost-effective method for the monitoring of MTX in osteosarcoma patients under high-dose MTX treatment, thus contributing to more efficient treatment.