The quantification of free and total alkannins in 13 endemic Anatolian Alkanna species is described for the first time. Extraction of the samples was performed by sonication with hexane, followed by hydrolysis in 1 N NaOH. For analysis, a new HPLC method, utilizing reversed phase material (Synergi Max RP), was developed and successfully validated. The obtained data confirmed that the assay is sensitive (LOD of 13 ng on-column for alkannin), accurate (the recovery rate was 92.3%) and precise (RSD <= 6.8% for intra-day precision; RSD <= 4.8% for inter-day precision). LCMS experiments were performed to assure peak identity and purity. Quantitative results revealed that roots of Alkanna tubulosa showed the highest amount of total alkannins (0.84%), while Alkanna mughlae contained the highest percentage of free alkannins (0.04%). Compared to the commonly used A. tinctoria (total alkannins: 0.11 to 0.54%; free alkannins: 0.002 to 0.01%) these two species could therefore serve as alternative sources for medicinally important alkannin derivatives.