The Jian-er syrup is a commonly used traditional Chinese medicinal preparation refined from two herbs, Metaplexis japonica (Thunb.) Makino and Justicia procumbens L. A new lignan, named 6′-hydroxy-Justicidin B, was isolated from the ethyl acetate extract of the Jian-er syrup and its structure was established on the basis of spectral analysis. The content of 6′-hydroxy-Justicidin B in the Jian-er syrup was determined by RP-HPLC for the first time. Chromatographic separation was achieved on an Agilent Zorbax SB-C18 column (150 mm × 4.6 mm i.d., 5 μm) at 25 °C and the mobile phase was a mixture of acetonitrile and water (31:69 v/v) detected at 256 nm at a flow rate of 1.0 ml/min. This method was validated in terms of selectivity, linearity, precision, accuracy, limit of detection, limit of quantitation and solution stability. It can be used to control the quality of this preparation effectively.