Tropane alkaloids, recognized for their significant therapeutic applications, are primarily derived from plant species such as Datura, Duboisia, Atropa, Hyoscyamus, Schizanthus, and Anisodus. These alkaloids are extensively used in the pharmaceutical and herbal industries for their anticholinergic and other medicinal properties. However, the large-scale cultivation of these plants to meet current demand is hindered by substantial variability in alkaloid content across harvested materials, posing challenges to consistent production. The growing global demand for tropane alkaloids and the difficulties of achieving uniform yields have underscored the need for alternative approaches. Biotechnological advancements, particularly micropropagation, offer promising solutions for the sustainable production of these valuable compounds. This chapter provides a detailed exploration of micropropagation techniques tailored for major tropane alkaloid-yielding plants. It elaborates on the selection and preparation of explants, highlighting sterilization protocols to ensure contamination-free cultures. Comprehensive information on the composition of culture media, the role of plant growth regulators, and optimized concentrations for effective shoot and root induction is presented. The chapter also discusses the combination of plant growth regulators and their impact on regeneration efficiency. Additionally, procedures for acclimatizing in vitro-propagated plantlets to ex vitro conditions are described. By addressing these critical aspects, this chapter emphasizes the potential of micropropagation to enhance alkaloid production and contribute to the conservation and large-scale cultivation of these medicinally important plant species.