An in vitro two-liquid-phase suspension culture system of Alkanna orientalis was established in order to evaluate the ability of cultured cells to produce naphthoquinone derivatives. Initially, callus tissues were prepared from cotyledon explants of A. orientalis on the solidified B5 medium supplemented with 1 mg/L 2,4-dicholorophenoxyacetic acid (2,4-D) and 2 mg/L kinetin (kin). Subsequently, fragile calli were transferred to liquid B5 medium with the same growth regulators for the initiation of cell suspension culture. Thereafter, the two-liquid-phase suspension culture was established using M9 medium and liquid paraffin as the absorbent. The presence of liquid paraffin efficiently induced the production of pigments by the cultured cells. The n-hexane extract of proliferated cell suspension culture was studied by analytical HPLC. Then, the main secondary metabolites were separated by preparative HPLC and their structures were elucidated by UV, 1H and 13C-NMR spectroscopy. Accordingly, alkannin/shikonin, alkannin/shikonin acetate, deoxyshikonofuran and a dimmer of alkannin/shikonin were identified as the main chemical compounds in the extract. On the basis of our results, the two-liquid-phase suspension culture led to the effective induction of naphthoquinone derivatives. These findings draw attention to the potential of A. orientalis cell suspension cultures in the production of naphthoquinone which may be considered as a source for the biosynthesis of these secondary metabolites for medicinal or industrial uses.