Plumbago becomes one of the essential medicinal, and decorative plants belonging to the family plumbaginaceae due to the present of plumbagin a secondary metabolite. In this study, the nodal segments of shoots of Plumbago auriculata were used as an explant in establishing in vitro cultures.  In vitro callus initiation and shoot regeneration was studied using mixtures of auxins, antiauxins (p-Chlorophenoxyisobutyric acid and 2,3,5-triiodobenzoic acid), and cytokinins. The medium comprising one mg/l PCIB and 0.5 mg/l BA produced the most significant outcomes in the case of callus induction. However, the best shooting number was given (11.18) with the same combination after eight weeks. According to the data, the leaves extract had the highest level of polyphenols (742.6 mg/100g.d. w) and flavonoids (563.9 mg/100g.d. w).