AbstractMethotrexate is a common component of pediatric oncology treatment and delayed clearance increases risk of significant toxicities. Glucarpidase is indicated for patients with toxic plasma methotrexate concentrations with renal toxicity. Laboratory interference with immunoassay measurement post‐glucarpidase administration is well established, with current product labeling indicating this persists for 48 h. However, recent experience in pediatric patients supports this discrepancy persists beyond 48 h. Three cases experienced delayed methotrexate clearance and received glucarpidase with subsequent measurement of methotrexate levels by liquid chromatography tandem mass spectrometry (LC–MS/MS) and/or immunoassay. Within this case series, discrepancies between LC–MS/MS and immunoassay levels persisted significantly longer than 48 h.