Isohexenylnaphthazarins (IHN), commonly known as Alkannins and Shikonins (A/S), are lipophilic red pigments. They are found in the underground parts, mainly roots, of at least a hundred and fifty species that belong to the genus Alkanna, Lithospermum, Echium, Onosma, Anchusa and Cynoglossum of the Boraginaceae family. The chiral pair A/S are potent pharmaceutical substances with a well-established and wide spectrum of wound healing, antimicrobial, anti-inflammatory, antioxidant, anticancer, radical scavenging and antithrombotic biological activity. The last years there has been extensive scientific research in many areas throughout the disciplines of chemistry and biology and more specifically in cancer chemotherapy and a number of papers have appeared in the literature. Significant research has been conducted on A/S effectiveness on several tumors and on their mechanism of anticancer action. A/S and their derivatives are susceptible to several transformations, such as photochemical decomposition, thermal degradation and polymerization. The stability of these substances during processing and storage is crucial to their use in pharmaceuticals and cosmetics, since polymerization of A/S results in a reduction in their antimicrobial activity, decrease in concentration of the active monomeric ones and to limited applications, due to loss of deep red colour and a decrease in solubility. Therefore, the determination of the impurities, degradation products or byproducts with the use of several analytical techniques, is of great importance. Additionally, the identification, qualitative and quantitative determination of A/S and their derivatives in raw materials for pharmaceuticals, such as natural products, samples prepared either by plant tissue cultures, or synthetically or by hydrolysis of naturally occurring IHN esters, is crucial for their use in pharmaceuticals. A large number of analytical techniques have been applied for the analysis and study of alkannin, shikonin and their derivatives. Chromatographic techniques used include TLC densitometry, Size Exclusion Chromatography, HPLC and chiral HPLC. The detection and identification techniques include: UV/Vis, IR, FTIR, 1H 1D and 2D NMR, 13C-NMR, mass spectrometry, FAB-MS, MALDI-MS, circular dichroism and indirect atomic absorption. Polarography, voltammetry, differential pulse voltammetry and even a photoacoustic technique for transdermal adsorption measurements have been utilized for qualitative and quantitative determinations of A/S and their derivatives. In the present study, all the above mentioned analytical methods on alkannins and shikonins are reviewed.