OBJECTIVE：To establish a method for the content determination of L-shikonin in Zilian liniment and evaluate the uncertainty by 2 calculation methods. METHODS：HPLC was performed on the column of Waters C18 with mobile phase of methanol- 0.025 mol/L phosphate（85 ∶15，V/V）at flow rate of 1.0 ml/min，the detection wavelength was 516 nm，column temperature was room temperature，and the injection volume was 10 μl. Quantitative analysis was conducted for the process and procedures calculated by standard curve method and external standard method，and the uncertainty was evaluated. RESULTS：The linear range of L-shikonin was 4.024-80.48 μg/ml；RSDs of precision，stability and reproducibility test were lower than 2.0％；recovery was 88.5％-93.1％ （RSD＝2.0％，n＝6）. Standard curve method was used to determine the content，and the expanded uncertainty was 0.70 μg/ml；external standard method was used to determine the content，and the expanded uncertainty was 0.30 μg/ml. CONCLUSIONS：Standard curve method is more economical，more convenient and lower uncertainty in the content determination of L-shikonin in Zilian liniment.