Objectives: Dried roots of Echium species are used in Turkey for the treatment of wounds, inflammation, and depression. In this study, an reversed-phase-liquid chromatographic method with isocratic elution was developed to determine shikonin derivatives in Echium species. Materials and Methods: The chromatographic separation and quantification was performed on a C18 column (ACE, 150 mm × 4.6 mm, 5 μm), with a mobile phase of acetonitrile and 0.1 M acetic acid (70: 30, v/v) at a flow rate of 1 mL/min, and ultraviolet detection at 520 nm. Results: Linear behavior was observed over the investigated concentration range (2-500 ppm) for all analytes, with a correlation coefficient of >0.998. The proposed method was found to be specific and precise for the quantification of shikonin derivatives in Echium species. Conclusion: The highest content of shikonin derivatives was observed in E. italicum L. compared with the other species studied herein, advocating the use of E. italicum L. roots as an alternate source for shikonin derivatives.