Abstract: A simple, sensitive and accurate method for the separation and determination of the naphthoquinones, deoxyshikonin (Ⅰ), acetylshikonin (Ⅱ) and β-β-dimethylacrylshikonin (Ⅲ) in tissue cultured Zicao is described. The sample solution was applied at a point 1 cm from the bottom edge of the TLC silica gel plate ( 10×15cm ). A mixture of n-hexane- acetone- chloroform-glacial acetic acid ( 13:0.3:0.2:0.05 ⅴ/ⅴ) was used as the eveloping solvent. The plate was saturated for 30 min and then devdoped twice for 15 cm using ascending technique. The spots were scraped off and extracted with CHCl₃, then determined spectrophotometrically.The standard curve was linear in the range of 0.65～2.25μg for the naphthoquinones. The method has been applied to the analysis of different samples. This method can be used for the quality control of naphthoquinones in tissue culture products.