This chapter emphasizes the challenges of extracting tropane alkaloids from their natural sources and the need for alternative methods, such as in vitro synthesis, to meet the growing demand. It covers the advancements in in vitro synthesis and other production techniques like cell cultures, organ cultures, and genetic engineering. Further, this chapter evaluates the advantages and disadvantages of each approach and provides insight into the current state of tropane alkaloid synthesis in vitro. It also discusses the factors affecting tropane alkaloid synthesis, including precursor availability, culture conditions, and biotechnological interventions. Additionally, it covers optimization strategies such as medium composition, elicitation techniques, and bioreactor systems to enhance alkaloid yields and productivity.