An important medicinal plant, Pueraria tuberosa (Roxb. Willd DC), is a perennial woody climber in the Fabaceae family. The current study aims to develop callus from the leaves and nodes of Pueraria tuberosa as well as to optimize the in vitro regeneration method for Pueraria tuberosa. Murashige and Skoog's medium supplemented with Benzlyaminopurine (4.44 µM) and naphthalic acetic acid (2.68 µM) is more effective for leaf and node callus. The nodal explant was cultured in Murashige and Skoog medium supplemented with (BAP+NAA) (0.66+0.52) µM to achieve direct shooting from nodal segments. Shoot multiplication was significantly influenced by the type and concentration of plant growth regulators as well as by subculturing in vitro regenerated shoot lets on fresh media. Transferring in vitro shoots to rooting media containing 2.68 µM NAA and 0.49 µM indole butyric acid (IBA) resulted in the growth of healthy roots. An efficiency of 80% was achieved during the well-rooted plantlet's acclimatization. This study compares the phytochemical content of P. tuberosa plants that grew in the wild and those that were developed in vitro. In vivo and in vitro qualitative and quantitative phytochemical investigation revealed that they are a valuable source of secondary metabolites. In a conclusion, the present study will be helpful in the herbal formulations that use any in vitro parts of P. tuberosa without destroying plants in wild conditions. Additionally, the work will aid in the conservation of this rare medicinal plant.