Pralatrexate (PDX) is a folate antagonist structurally similar to methotrexate (MTX). Unlike MTX, it is currently not known whether PDX exhibits delayed clearance and heightened toxicity in the setting of fluid overload. A specific serum assay for PDX is not commercially available. To our knowledge, we report the first case using an MTX serum assay as a surrogate for PDX concentrations to avoid a potential drug-drug interaction with pralatrexate. We describe a 76-year-old man with refractory cutaneous T-cell lymphoma who began therapy with weekly PDX 15 mg/m2 intravenous infusions on days 1, 8, and 15 of a 28-day cycle. He subsequently developed mucositis, a moderate right-sided pleural effusion, and peripheral edema over the next 5 weeks. Aggressive diuresis with furosemide was initiated, which was then withheld the day before his next PDX dose to avoid a potential drug-drug interaction between PDX and furosemide. His baseline MTX/PDX concentration (measured prior to administration of the cycle 2, week 2 PDX dose) was less than 0.20 μmol/L (i.e., undetectable). After PDX administration, his 1-hour peak MTX/PDX concentration increased to 0.58 μmol/L. Aggressive diuresis was withheld until his MTX/PDX concentration was undetectable, 43.5 hours later. PDX is more potent than MTX and displays similar pharmacokinetic properties. PDX concentrations using the serum MTX assay reflect lower values than those reported from PDX-specific assays in clinical studies. Because PDX is approved by the U.S. Food and Drug Administration for the treatment of uncommon malignancies, it is unlikely that a specific assay will be commercially developed. We propose that the MTX serum assay has merit for use in determining when to reinstate possible interacting drug therapies such as loop diuretics.