A rapid, simple and sensitive reversed-phase HPLC method with Ultraviolet (UV) absorbance measurement at 280 nm was applied for the simultaneous quantitative determination of marker substances in the traditional Chinese herbal ointment Shiunko. Shiunko consists of Lithospermum radix containing shikonin, acetylshikonin, β,β dimethylacrylshikonin, isobutylshikonin, deoxyshikonin, and isovalerylshikonin, as well as Angelica radix containing ferulic acid. Specimens of Shiunko ointment were extracted with acetonitrile, separated on a reversed-phase C18 (ThermoQuest, 250 × 4.6 mm i.d.) column at ambient temperature, with a solvent system consisting of 47% acetonitrile and 53% acetic acid (v/v, pH 2.56) at a flow rate of 1.0 mL/min. Diclofenac was used as the internal standard. This procedure enabled a run time of within 35 min. In the validation of this analytical method, the RSD and RE were less than 7.0%, indicating satisfactory accuracy and repeatability. The linear relationships between the concentration of analyte in samples within a given range (ferulic acid 0.175- 3.50 μg/mL, shikonin 0.5-10.0 μg/mL, acetylshikonin 0.5-10 μg/mL, β,β-dimethylacrylshikonin 0.35-7 μg/mL, isobutylshikonin 0.4-0.8 μg/mL, deoxyshikonin 0.255-5.1 μg/mL, and isovalerylshikonin 0.25-5 μg/mL) and the corresponding peak area ratio were good indications that this analytical method could be used in quantitative analysis. The lower limit of detection of these marker substances under above conditions was 0.05 μg/mL.