This work reports the extraction procedures of alkannin/shikonin mixture from roots of six populations of Onosma echioides, by means of three extraction techniques: Soxhlet extraction, maceration and rapid solid-liquid dynamic extraction (RSLDE). Five solvents with different polarity (hexane, petroleum ether, chloroform, ethyl acetate, methanol) were also studied. Analysis of the extracts was performed by an HPLC-DAD (diode array detector) system. The most efficient extraction technique was Soxhlet procedure using ethyl acetate for 6 h. Studied samples of O. echioides showed an alkannin/shikonin content in the range of 0.02-0.24 mg/kg. Other naphthoquinone derivatives (deoxyalkannin/deoxyshikonin and 5,8-dihydroxy-2-(4-methyl-6-oxo-5,6-dihydro-2H-pyran-2-yl)-[1,4]naphthoquinone and arnebin-6) were found for the first time in O. echioides and characterized in the extracts using HPLC-MS apparatus equipped with an ESI ionization source.