Lithospermi Radix has been used in traditional medicine for antimicrobial, antiinflammatory, immunostimulating, antitumor and wound-healing activities. For the quality control of this drug, acetylshikonin was isolated from the hexane extract of Lithospermum erythrorhizon(Boraginaceae) and identified by the spectroscopic evidences. A quantitative analysis of acetylshikonin using HPLC method showed that the average contents were $0.084{pm}0.026%$ in 31 samples collected throughout the various regions of Korea.