Investigations were undertaken to explore a successful and reproducible protocol for plant regeneration of Lawsonia inermis L. Shoot tip and nodal segment were excised and cultured upon MS medium supplemented with various cytokinines i. e. BAP, kinetin, zeatin, GA3 and spermidine. The explant responded successfully at a concentration of BAP (2 mg/l). Additives like silver nitrate (AgNO3), adenine sulphate (AdSO4) and casein hydrolyzate (CH) were tried for improving shoot regeneration abilities. A complete success was achieved using BAP (2 mg/l) along with kinetin (0.25 mg/l) and adenine sulphate (20 mg/l) after 45 days. For root induction, surgically separated microshoots were cultured upon half strength MS medium containing 0.5 mg/l indole-3-butyric acid (IBA) with an average of 76.66%. After one month, the in vitro regenerated plantlets were transferred to greenhouse condition in clay pots containing a mixture of sand: soil: vermicompost (1: 1: 1). The regenerated plantlets were subjected to GC-MS analysis and compared with the mother explants. The higher beta-glucopyraoside, methyl (59.75%) and alpha linoleic acid (11.25%) were observed indicating the application of plantlet in skin aliments and heart problems. The increase in active phytochemical components of in vitro regenerated plants over its mother plants showed the applicability of this technique over conventional method.