The anodic voltammetric behavior of methotrexate was studied at glassy carbon electrode in acetate buffer (pH = 3.6) solution using cyclic, square-wave voltammetric and chronocoulometric techniques. The oxidation of methotrexate is an irreversible diffusion-controlled process. The oxidation mechanism was proposed and discussed in this work. The dependence of the current on pH, the concentration and nature of buffer, and instrumental parameters were investigated to optimize the experimental conditions for the determination of methotrexate. It was found that in the range of 8.0 x 10(-7) -2.0 X 10(-5) mol/L, the currents. measured by square-wave voltammetry presented a good linear property as a function of the concentrations of methotrexate. In addition, validation parameters, such as reproducibility, sensitivity and recovery were evaluated as well. The proposed method was also successfully applied for the determination of methotrexate in diluted human urine with good satisfactory. (c) 2007 Elsevier B.V. All rights reserved.