Plant secondary metabolites are synthesized in response to specific environmental cues, such as defense against pathogens or adaptation to stress conditions, while primary metabolites dominate plant biosynthesis under normal circumstances. The underlying hypothesis guiding yield enhancement strategies in plant cell and tissue cultures is to simulate these natural stimuli within controlled environments, thereby prompting the production of secondary metabolites in desired quantities. These strategies, designed to mimic the biotic and abiotic factors that trigger secondary metabolite synthesis in vivo, aim to exploit the inherent biochemical responses of plants to induce metabolite production in vitro. Elicitation involves exposing plant cell cultures to stimuli like microbial elicitors or environmental stresses such as UV light or temperature fluctuations, effectively activating plant defense pathways and enhancing secondary metabolite synthesis. Similarly, precursor feeding supplements the culture medium with specific precursor molecules required for secondary metabolite biosynthesis, ensuring that metabolic pathways have ample substrates to drive higher production rates. Biotransformation harnesses the enzymatic capabilities of plant cells to convert simple substrates into complex secondary metabolites, offering a route to produce high-value compounds that may be challenging to synthesize chemically. Immobilization techniques, on the other hand, stabilize plant cells within a matrix, sustaining their metabolic activity and enhancing productivity by providing a controlled microenvironment. Despite their potential, these strategies come with challenges, including maintaining genetic stability, optimizing culture conditions, and addressing inherent limitations in metabolite productivity. Nonetheless, through careful optimization and integration of advanced biotechnological approaches such as metabolic engineering and synthetic biology, yield enhancement strategies hold promises for enabling sustainable and efficient production of secondary metabolites in plant tissue cultures, meeting the growing demands across various industries. This chapter provides a comprehensive overview of the strategies and methodologies for enhancing the yield of secondary metabolites in vitro, featuring their importance in both scientific research and practical applications.