A procedure for the determination of rifampicin by adsorptive stripping voltammetry (AdSV) has been optimized. Two different voltammetric techniques, namely differential pulse adsorptive stripping voltammetry (DPAdSV) and square-wave adsorptive stripping voltammetry (SWAdSV), were used. In order to determine the most sensitive technique, an optimization of the experimental parameters was carried out for each one using experimental design methodology. The detection limits obtained, 6.135×10−9 mol dm−3 for DPAdSV and 9.828×10−9 mol dm−3 for SWAdSV, show that DPAdSV is a more sensitive technique for the analysis of rifampicin. Finally, this method was used to determine rifampicin in pharmaceutical preparations and urine samples.