A simple high performance liquid chromatographic (HPLC) method for the determination of 2-diazonaphthoquinone-5-sulphonic acid phenyl ester (DNQ) is developed. 5-Amino naphthalene-1-sulphonic acid sodium salt is used as internal standard. A Polaris C18-5 mu silica column (250x4.6 mm), 5 mu particle size, was equilibrated with a mobile phase composed of methanol and water (80:20, v/v). Flow rate was 1.0 mL/min. The elution time for 5-amino naphthalene-1-sulphonic acid sodium salt and DNQ was approximately 2 and 4 min, respectively. Calibration curve of DNQ was linear in the concentration range of 10-50 mu g/mL. Limit of detection and quantification were measured in the lowest concentrations of the replicates. Recovery is about 96%.