Asparagus officinalis as a valuable medicinal plant has a low multiplication rate using the conventional methods. This study was carried out to establish an efficient in vitro propagation protocol and also to compare some biological activities of in vivo and in vitro grown Asparagus. The nodal explants were cultured on MS medium supplemented with different concentrations of 6-benzylaminopurine (BAP) and 1-Naphthaleneacetic acid (NAA) or kinetin (Kn) and Indolebutyric acid (IBA), under light and dark conditions. After 6 weeks of culture, the highest percentage (100%) of callus formation was found in 17 of treatments under dark condition and 3 treatments under light condition. Also between the two groups of hormones, Kn +IBA showed better results in promoting callus formation. The highest average number of shoots (4.25) of size 4 mm or more per explant, formed under dark condition using 1.5 mg/L BAP mixed with 0.05 mg/L NAA. Rooting was best induced in shoots excised from shoot cultures which were proliferated on MS medium supplemented with an optimal concentration of 0.4 mg/L IBA (2 roots per explant). In the second part of the study, the extracts of in vivo and in vitro grown plants as well as callus tissue were tested for their total phenolic and flavonoid content, antioxidant and antityrosinase activities, using two different extraction solvents (methanol and hexane). The methanol extract of in vivo grown plants showed a significantly higher amount of total phenolic and flavonoid content. The antioxidant activity of tested samples followed this order; in vivo plant > callus > in vitro plant.