An in vitro cell suspension culture of Echium italicum was established and assayed for the production of shikonin and alkannin derivatives. Callus tissues were induced from cotyledon explants of the plant incubated onto the solidified B5 medium. A two-liquid-phase system suspension culture was then established to elicit pigments of shikonin and alkannin derivatives using liquid paraffin. The presence of liquid paraffin efficiently induced pro-duction of pigments in cultured cells. The production and/ or accumulation of these compounds in the E. italicum cells was examined using fluorescence microscopy as the naphthoquinone molecules display autofluorescent proper-ties. Phytochemical analysis of the n-hexane extract of the medium was also carried out using preparative HPLC. The chemical structure of shikonin and alkannin derivatives were characterized by UV, 1H-NMR, and 13C-NMR techniques. Based on our findings, this bioprocess engi-neering approach resulted in induction of shikonin and alkannin derivatives, whereupon it may be recruited for production of these important secondary metabolites.