Plant tissue culture can readily be applied to the field of medicinal plants and secondary metabolites production. In this study, in vitro propagation protocol of Rumex cyprius Murb. was established. The highest shoot multiplication rate was observed from explants grown on MS medium supplemented with 1.5 mg/L BA (7.8 shoots per explant). For rooting, the maximum root induction rate was observed on MS medium supplemented with 1.5 mg/L IBA or 1.0 mg/L NAA. The longest roots were observed for plants grown on MS medium supplemented with 1.5 mg/L IBA. For callus induction, leaves from in vitro grown plants were cultured in various levels of 2,4-D or different combinations of BA and NAA. The highest fresh weight (5.86 g) of calli was observed at 1.5 and 0.5 mg/L BA and NAA, respectively. The second part of this work investigated the effects of abiotic factors (NaCl and Mannitol) and biotic factors (Yeast extract and Chitosan) on secondary metabolites accumulation of in vitro grown R. cyprius. Phenolic compounds were extracted, and the individual phenolic compounds and antioxidant activity of the extracts were determined of in vitro grown R. cyprius. The total phenol content and antioxidant activity increased significantly by the pervious factors. Reversed high-pressure liquid chromatography (RP-HPLC) was used to measure the effect of these factors on the contents of individual phenolic compound. New phenolic compounds (Gallic acid and Chlorogenic acid) not found in R. cyprius plants grown on control media were produced in R. cyprius plants grown on media supplemented with elicitors. Results indicate that using elicitors in plant tissue culture could be used to produce/enhance secondary metabolites accumulation in R. cyprius.