Objective To establish the determination method of acetylshikonin in three species， Onosma paniculatum Bur. et Fr. ， O. cingulatum W. W. smith et J. E. Jeffrey and O. confertum W. W. Smith.. Methods Using HPLC as quantitative analysis means and acetylshikonin as the detection of targets， the quality standards of above three species were studied. Results The acetylshikonin had good linear relationship in the range of 0.0952～1.9030μg. Precision， stability， repeatability and recovery are achieved. Conclusion The method is simple， accurate， reproducible and suitable for determining the acetylshikonin in Onosma paniculatum and its two closely related species.