Abstract The objectives of this study were to develop and validate a method for the simultaneous HPLC-UV determination of obidoxime (OBI), scopolamine (SCA), Atropine (ATR) and imipramine (I) in an aqueous antidote solution. An ion-pair mechanism with DAD (diode array detector) detection was used to quantitatively determine the four drugs. The proposed HPLC method uses a Kromasil 100-5C8 (250x4.6mm) column, gradient elution, at 25°C column temperature.