A novel sensitive voltammetric method for the determination of an important chemotherapeutic drug, methotrexate, was designed using a bare boron‐doped diamond electrode in connection with differential pulse voltammetry. Methotrexate provided one well developed oxidation peak suitable for analytical purposes at about +1000 mV (vs. Ag/AgCl electrode) in an acidic medium. The voltammetric behavior of methotrexate in dependence of pH of the supporting electrolyte and scan rate has been investigated using cyclic voltammetry and DC voltammetry, respectively, to select the suitable experimental parameters. The optimum conditions for its determination using differential pulse voltammetry have been found to be 0.05 mol L−1 H2SO4 with a limit of detection of 1.0×10−8 mol L−1 and a linear dynamic range from 5×10−8 to 2×10−5 mol L−1. Calculated values of relative standard deviations of repeated determinations (n≥5) in various concentration levels of methotrexate did not exceed 4 %. The influence of possible interfering agents was studied as well. Finally, practical applicability of the newly proposed method was verified by analysis of real drug preparations and spiked human urine. The calculated recovery values ranged from 99.2 to 101.4 %.