In normal and malignant human cells, the folate antagonist methotrexate (MTX) is converted to a series of polyglutamates (MTXGlu n , n=2–5) which play a role in its therapeutic efficacy. Here we report an assay to determine MTX and MTXGlu n in Caco-2 cells exposed to MTX. After a simple protein precipitation step, cell homogenates (2×10 6 cells) were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) using aminopterin as internal standard. Separation was by reversed phase HPLC on a C8 column using gradient elution with 0.1% formic acid and acetonitrile. Detection was by electrospray ionization in the positive ion mode followed by multiple reaction monitoring of the transitions of the [M+H] + ions of MTX and MTXGlu n to their common product ion at m/z 308.2 and of aminopterin at m/z 441.3→294.2. Calibration curves for all analytes were linear in the range 2–250nM (r 2 >0.996). Intra- and inter-day precisions (as coefficient of variation) were 3.4–15.1% and 4.3–18.4%, respectively with corresponding accuracies (as relative error) of −3.6 to +6.6% and −5.5 to +7.5%, respectively. Recoveries were in the range 60±4 to 108±13%. It was found that MTX undergoes only limited polyglutamation in Caco-2 cells exposed to MTX over 24h.