This chapter explores the integrated use of elicitation and biotransformation in hairy root cultures as a sustainable and efficient biotechnological strategy for enhancing the production of alkaloids bioactive secondary metabolites with significant pharmaceutical, agricultural, and industrial relevance. Elicitation, through the application of biotic and abiotic stressors, activates plant defence pathways and stimulates the biosynthesis of target alkaloids. Biotransformation complements this process by enabling the structural modification of precursor compounds into high-value or novel alkaloid derivatives using specific enzymatic machinery. The chapter provides a comprehensive overview of different elicitor types, biosynthetic pathways, precursor feeding strategies, and in vitro culture systems on medicinal plants. Additionally, it highlights the key challenges in scaling up hairy root-based production, such as genetic stability, contamination, and cost of inputs, while outlining recent innovations that address these issues. By presenting both foundational knowledge and current advancements, the chapter underscores the transformative potential of elicitation and biotransformation to revolutionize natural product synthesis and pave the way for industrial-scale, plant-based pharmaceutical production.