To enable effective quality control of the traditional Chinese medicine (TCM) preparation the Wuji pill, seven bioactive components, i.e. paeoniflorin, jatrorrhizine, coptisine, palmatine, berberine, evodiamine and rutaecarpine, from the three medicinal herbs composing Wuji pills have been selected as evaluation markers. Then an ultra-performance liquid chromatography (UPLC) method coupled with photodiode array (PDA) detection was developed and validated for simultaneous quantification of the 7 mark components in the Wuji pill. Optimum separation was achieved on a C₁₈ column (50 × 2.1 mm i.d., 1.7 μm particle) through a 5.0 min gradient delivery of a mixture of A (acetonitrile) and B (0.1% aqueous phosphoric acid containing 1.38 mmol L⁻¹ sodium dodecyl sulphonate (SDS) and 5% acetonitrile (v/v)) at a flow rate of 0.3 mL min⁻¹ at 25 °C. Because of the different UV characteristics of these components, 3 detection wavelengths were used for quantitative analysis. All of the analytes showed good linearity (r of >0.999). The limit of detection and limit of quantitation for the analytes ranged from 0.005 to 0.15 μg mL⁻¹ and from 0.01 to 0.50 μg mL⁻¹, respectively. The method was validated for repeatability, precision, stability, accuracy, and selectivity. The RSD values of retention time and peak area were 0–0.3% and 0.1–0.9% for intra-assay, and 0.1–1.4% and 1.0–2.6% for inter-assay precision. Analysis repeatability and stability (24 h) were all lower than 2.0%. The recovery of the method was 96.1–97.5% with a RSD of 1.1–2.8%. The validated method was applied to the quality control of Wuji pills from different production batches.