Phenylethanoid glycosides, the main constituents of the aerial part of eyebright (Euphrasia rostkoviana Hayne) were treated by the endogenous hydroxylase enzyme and the concomitant biotransformation was characterized by applying high-performance liquid chromatography with UV and MS detections and NMR spectroscopy. In the extracts of the untreated (intact) samples, acteoside and eukovoside were determined as main compounds. The enzymatic treatment resulted in the quantitative transformation of these phenylethanoid glycosides into their corresponding hydroxyl derivatives identified as two epimers of beta-hydroxyacteoside and beta-hydroxyeukovoside. As to the importance of this hydroxylation beta-hydroxyeukovoside was identified as a new compound and beta-hydroxyacteoside was described for the first time in eyebright. We proved for first time that a beta-hydroxylase enzyme is active in eyebright tissues which can transform phenylethanoid glycosides into their beta-hydroxyl derivatives. Our new enzymatic method combined with a preparative HPLC facilitates the isolation of beta-hydroxyl phenylethanoid glycosides from the aerial part of eyebright. (C) 2014 Elsevier Ltd. All rights reserved.