The investigations were targeted to obtain in vitro culture of Plantago ovata, which would be efficient in the production of plant biomass and phenolic compounds with multiple biological activities, like acteoside and plantamajoside - the main phenylethanoid glycosides present in the Plantago genus. In these experimental studies, roots were obtained by natural development from seedlings germinated in vitro and from in vitro multiplied shoots as well as by direct organogenesis on seedling leaf explants and indirect organogenesis from callus cultured on various explants. Callus-regenerated roots agitated in MS (Murashige and Skoog) liquid medium supplemented with phytohormones NAA (1-naphthaleneacetic acid) 0.25 mg/L and BAP (benzylaminopurine) 0.5 mg/L turned out to be a good growth system for root biomass production - mean increase of root fresh weight calculated on three following passages was 6641.07%. The quantitative screening of the root extract by HPTLC video densitometric method showed the content of acteoside 33.02 mg/g dry weight (d.w.) (538.9 mg/L of culture) and plantamajoside 6.39 mg/g d.w. (104.0 mg/L of culture) in root biomass obtained with the yield 16.32 g d.w. /L within 4 weeks. The content of those compounds in the in vitro shoots was significantly lower – 4.20 and 4.28 mg/g, d.w., respectively. Although the high level of acteoside in the root culture decreased in the following 8 weeks of the culture, it is still satisfactorily high (8.25 mg/g, d.w.). This is the first report on the adventitious root cultures of Plantago ovata and the content of those phenylethanoids in that well-grown plant material. The proposed approach provides an alternative to the soil-grown plants, source of plant metabolites, due to the possibility of the fast, regular and scalable production of plant biomass containing the compounds of interest as the main constituents.