A simple isocratic reverse-phase HPLC method for the determination of six antichagasic phenylethenylbenzofuroxans and its major synthetic secondary products, the corresponding geometric isomers and the benzofurazans, was developed and validated for use in the analysis of pre-clinical studies. Separation was achieved on a reverse-phase Supelco LC-18 column using either methanol-acetonitrile-water or acetonitrile-water, in different proportions, as mobile phase. The compounds were eluted isocratically at a flow rate of either 0.8 or 1.0 mLmin(-1). The compounds were analyzed with UV detection at 210 and 300 nm. The validation characteristics included linearity, accuracy, precision, specificity, limit of detection and quantification and robustness. Validation acceptance criteria were met in all cases. This method was used successfully for the quality assessment of the drugs production in the scale-up procedures.