Direct plant regeneration from different explants, micropropagation and determination of secondary metabolites were studied in the critically endangered endemic Rhaponticoides mykalea (Hub.-Mor.) M.V. Agab & Greuter. Seed germination was achieved by damaging the seed coat and cultivating the embryos on Woody Plant Medium (WPM), of which 40% germinated. The epicotyls and cotyledonary petioles of seedlings were used as initial explants and direct shoot regeneration was obtained on WPM containing 2.22M 6-benzyladenine (BA). WPM medium supplemented with 2.22M BA and 4.92M indole-3-butyric acid (IBA) significantly improved the production of multiple shoots, resulting in an average of 5.6 shoots per explants. The highest rooting of shoots (35.6%) was observed with WPM medium containing 19.68M IBA with 990M putrescine. Plantlets with well-developed roots were transferred to soil and acclimatised within a plant growth chamber. Acclimatised plants showed 100% survival rate and remained healthy. As a part of our study, the content of secondary metabolites in three tissue culture regenerated lines were determined by HPLC analysis. Chlorogenic acid, Quercetin and scutellarin were confirmed secondary metabolites of R. mykalea.