Lawsonia inermis commonly known as Mehandi or Henna , belongs to family Lythraceae .The present work was carried out with the objectives to devise suitable media for culture initiation , indirect organogenesis and biochemical estimations of callus induction during differentiation . Murashige and Skoog (1962) medium was used for the present investigation .This medium was fortified with various amount of auxins like Indole acetic acid , Napthalene acetic acid , 2,4-Dichlorophenoxy acetic acid (0.5,1.0,1.5,2.0 and 3.0 mg/l) and cytokinins like Kinetin and Benzylamino purine (0.5,1.0,1.5,2.0 and 3.0mg/l) individually and in various combinations .The medium containing , Dichlorophenoxy acetic acid resulted in highest percent induction of callus .There was no callus induction from internodal segments on medium devoid of growth regulators. Maximum percent callus induction upon cytokinin supplemented media was on BAP (3.0). The higher growth value callus developed from internodal segments was used for biochemical studies .There was decline in the contents of starch whereas the total soluble sugars , reducing sugars and total soluble proteins got increased .The activity of the enzyme alpha amylase of callus got increased and the activity of other enzymes i e. acid invertase is and acid peroxidase got decreased with the passage of time . UPLC analysis of extracts revealed that lawsone (naphthoquinone) was found in mother plant , regenerated plants and calli derived from intermodal segments of Lawsonia inermis. Highest lawsone content yield (0.154 µg /mg ) was observed in leaves of plant tissue culture raised plants with as compared to mother plant and the calli.