Isohexenylnaphthazarins (IHN), commonly known as alkannins and shikonins (A/S), are potent pharmaceutical substances with a wide spectrum of wound healing, antimicrobial, anti-inflammatory, and antitumor activity. Purification of A/S is crucial for their use in pharmaceuticals and for biological experimentation. Dimeric and oligomeric A/S derivatives co-exist with the active monomeric ones in most of the samples produced either by (semi)-synthesis or biotechnologically or isolated from natural products. Oligomeric A/S derivatives have not been studied for biological activity hitherto and a method to isolate them is essential.In the present study, solid-phase extraction (SPE) was applied for purification of commercial samples and isolation of monomeric and oligomeric A/S fractions, testing several stationary phases. Sephadex LH-20 cartridges achieved efficient purification for commercial samples containing both monomeric and dimeric A/S derivatives and also separation and isolation of both pure monomeric and dimeric A/S fractions for biological experiments. A high-performance liquid chromatography-diode array detection method was applied for detection, identification and quantification of monomeric and oligomeric shikonin fractions.