A voltammetric procedure optimized by experimental design for glyphosate determination in soil, water and vegetable samples is described. The voltammetry experiments were performed using a hanging mercury drop electrode (HMDE). The DPV variables involved in the optimization process were: voltage step, pulse amplitude, pulse interval, voltage step time and concentration of the supporting electrolyte. A full 2 5 factorial design was chosen to evaluate these effects. From the results obtained by the factorial design the three most important factors were determined. These variables were evaluated with a central composite design. Under the optimized conditions, the operational range was from 0.050 to 100.0 mg dm-3 and the detection and quantification limits were 14 and 48 µg dm-3 , respectively. The optimized method was successfully applied to glyphosate determination in soil, water and vegetables after purifying with an ion exchange resin and derivation.