An efficient in vitro callus induction and plant regeneration Protocol has been established for a value aromatic and medicinal plant, Aloysia triphylla by using shoots tips and leaf disc as explants. The nutrient MS medium with different concentrations of auxins NAA, IBA and 2,4-D alone or in combination with different cytokinins BA, TDZ and Kin were used to induce callus formation. Shoots tips and leaf disc cultured on MS medium supplemented with 2,4-D at 1.5 mg/L in combination with BA at 1 mg/L induced Maximum callus formation percentage of 91.7 and 100 %, respectively coupled with the highest callus fresh weight of 9.71 and 10.66 g, respectively. For Callus differentiation, callus derived from shoot tip and culture on half strength MS media fortified with TDZ at 2 mg/L and GA₃ at 0.5 mg/L recorded 100 % callus differentiation percentage and significantly the highest value of shoots number (12.92 shoots). Healthy regenerated shoots were rooted in vitro on MS containing 0.5 mg/L IBA. Plantlets with well-developed root and shoot systems were successfully acclimated (97%) and established in pots containing mixture of soil, sand and peatmoss (1: 1: 1).