A simple and specific method was validated for quantification of boldine in fluid extract of boldo (Peumus boldus Mol.) using high-performance liquid chromatography. A reversed-phase C₁₈ , Phenomenex® (150 x 4.6 mm, 4 µm) column was employed. The mobile phase consisted of 0.1 % trifluoroacetic acid and acetonitrile (78:22, v/v) at a flow rate of 0.8 mL/min. The column was maintained at 30 °C and the boldine peak detection was performed at a wavelength of 281 nm. The parameters used in the validation process were: linearity, specificity, precision, accuracy, limit of detection, limit of quantification and robustness. The validated method was selective and linear (r≥0.9991) for boldine concentration considering 5.0, 10.0, 15.0, 20.0 and 25.0 micro;g/mL. The recovery ranged from 90.93 % to 96.24 % and the limit of quantification was 2.41 micro;g/mL. The precision determined was reported as RSD (1.73 %). The method can be successfully applied to measure boldine concentrations in Boldo extract and be included in routine analysis of quality control.Colegio de Farmacéuticos de la Provincia de Buenos Aires