The family Verbenaceae includes 36 genera and 1035 species. Among them lemon verbena (Aloysia triphylla) is known to have high medicinal value. Therefore, development of fast and new in vitro micropropagation protocol will have a high importance in lemon verbena mass-propagation. This research study targeted to develop rapid in vitro micropropagation protocol for lemon verbena. Up to 88% of clean survived plantlets were obtained after treating the nodal explants with 0.5% berekina (NaClO) for 10 min. Shoot initiation and multiplication was achieved using node as explant planted on MS medium supplemented with different strength of 6-benzyladeninepourine (BAP) and kinetin (Kin) individually and in combination. Plants were put on root induction ½ strength MS medium fortified with different strength of indole-3-butyric acid (IBA) alone. The best treatment for shoot initiation was 6-benzyladeninepourine (1.5 mg/L) with 84.1% of initiation. The best treatment for shoot multiplication was 6-benzyladeninepourine (2.0 mg/L) with 9.23 shoots per explant. Best rooting (100%) and maximum root number per shoot (14.4) were found at 1.0 mg/L indole-3-butyric acid (IBA). The longest root (3.1 cm) was achieved without supplementing the media with plant growth regulators. The plantlets were hardened and acclimatized in fully automated greenhouse and survival percentage was greater than 70% planting on a combination of sterilized river sand, top forest soil and animal manure in a 1:2:1 (v/v/v) ratio. This in vitro micropropagation protocol can be used instead of conventional propagation techniques, as a fast and economically cheap method to propagate a wide range of similar plants.