Nowadays, high performance liquid chromatography (HPLC) represents an unavoidable step in the analysis of bioactive composition of plant substrates. However, there is a need for the development of a unique and universal method which would enable rapid, reliable and routine analysis of a large number of structurally diverse bioactive compounds from botanically different samples, such as medicinal plant extracts. Therefore, the aim of this study was to develop a unique HPLC-DAD method for determination of polyphenolic compounds in certain medicinal plants. In addition to the development and validation of HPLC-DAD method, spectrophotometric analysis of polyphenolic compound from aqueous extracts of sixteen medicinal plant species was carried out. The highest content of total polyphenols, hydroxycinnamic acids, flavones and flavonoids and the most potent antioxidant capacity were determined in extracts of the Lamiaceae plants (peppermint, lemon balm). The values of the limit of detection and quantification and high values determined for the accuracy and precision show the suitability of the developed HPLC method for the quantification of polyphenolic compounds in the evaluated plant species. The newly developed HPLC method in the studied plant species enabled the separation and determination of 24 polyphenolic compounds belonging to the hydroxybenzoic and hydroxycinnamic acids and flavonoids. By employing the developed method, high contents of rosmarinic acid and luteolin, characteristic for the plants of Lamiaceae family were determined, as well as individual polyphenolic compounds identified in only one plant species, which can be considered as a kind of their taxonomic biomarkers.