Artemisia ludoviciana Nutt. is an important aromatic plant widely used in traditional Mexican medicine for its therapeutic potential. Its medicinal activity is attributed to a wide range of bioactive compounds, including flavonoids. However, overexploitation, habitat loss, climate change and plant diseases threaten its natural populations and diversity. Although the species reproduces both sexually and asexually, conventional propagation methods are often slow, limited by environmental factors, and susceptible to pests and pathogens. Therefore, this research aimed to establish a micropropagation protocol for A. ludoviciana and to evaluate the changes in its phenolic composition and antioxidant activity while adapting to ex vitro conditions. Full-strength Murashige and Skoog (MS) media supplemented with 0.1 mg L⁻¹ 6-benzylaminopurine resulted in the highest number of shoots (3.30 ± 0.34) and shoot length (3.00 ± 0.12 cm). Moreover, 1/2 MS media supplemented with 0.5 mg L⁻¹ indole-3-acetic acid improved the number (14.45 ± 0.56) and quality of roots. Hardening and acclimatization of plantlets showed 100% survival after 10 weeks. Also, the phenolic composition and antioxidant activity of A. ludoviciana changed in response to stress derived from growth conditions. The results support the sustainable use and rapid propagation of the species, as well as provide the basis for the study of secondary metabolism in the plant.