Medications that have a narrow therapeutic window must be regularly monitored to assist in clinical dosing. Methotrexate (MTX) is one such chemotherapeutic agent that is closely monitored using various standard assays. Patients who have renal failure and who are treated using high-dose protocols are often given carboxypeptidase-G2 (CPDG(2)) as a chemoprotective agent. In this setting an inactive metabolite of MTX, 2, 4-diamino-N(10)-methylpteroic acid (DAMPA) is produced. DAMPA cross-reacts with MTX in most immunoassays, thus making them unsuitable for the monitoring of MTX in the setting of CPDG(2) therapy. We describe a rapid LC-MS-MS method that can be used to determine MTX levels in these cases.