In recent years, xanthones have received considerable attention from scientists due to their biological activity: anticarcinogenic, antiviral, antibacterial, antioxidant, anti-inflammatory and other properties.Therefore they are useful for prevention and treatment of different diseases:cancer, Alzheimer's and Parkinson's disease, cardiovascular disorders, diabetes, etc. Extracts of different species of plants containing xanthones are components of chemotherapeutic and other medical drugs. In order to find the most sensitive and environmentally safe method of quantitative determination of xanthones in the plant material and the nutrient medium, known methods were tested and selected for the prototype Vyisochina G. I. et al., 2011 method, which uses ethanol as an extractor. As the plant material we used plants of different species that were grown under in vitro cultivation conditions on the agarized nutrient medium. This agarized nutrient medium was also used for the xanthone content analysis. Based on the performed research, modifications of the method for determining the content of xanthones were adapted to the in vitro conditions, which detail the specificity of extraction and quantitative calculation of the xanthone content in plant explants. Our own method of determination of these compounds in the agarized nutrient medium was developed as well. The method, that we proposed, will significantly speed up the process of xanthone detecting and will also increase their yield in biotechnological processes for obtaining the pharmacologically valuable secondary metabolites of phenolic nature.