Methotrexate is an efficacious immunosuppressant for induction and maintenance of remission in Crohn's disease. The goal of this pilot study was to determine whether total or individual methotrexate glutamate levels (MTXGlu(n)) in red blood cells correlate with disease activity and adverse events in Crohn's disease. A cross-sectional study was undertaken with 12 patients on a stable dose of 25 mg weekly methotrexate (oral or subcutaneous). Clinical disease activity was assessed by the Harvey-Bradshaw Index (HBI), and biologic disease activity was measured by inflammatory markers. Concentrations of individual MTXGlu(n) levels were measured in red blood cells (RBCs) using high-performance liquid chromatography-mass spectrometry. No association was observed between RBC individual (MTXGlu(n)) or total methotrexate glutamate concentrations and clinical disease activity (HBI score) or inflammatory markers or adverse events. Although Crohn's disease patients in remission appeared to generally have higher RBC total longer-chain methotrexate polyglutamate (MTXGlu(3+4+5)) concentrations compared with those with active disease, a definitive association between RBC MTXGlu(3+4+5) levels and clinical disease activity could not be established. Larger longitudinal studies in patients with diverse disease activity are needed to establish the value of MTXGlu(n) levels as indicators of treatment efficacy and clinical outcome.