<p>Saponins are plant secondary metabolites synthesized through the triterpenoid biosynthetic pathway by a series of modifications, including acylation, glycosylation, and oxidation of β-amyrin, a product of 2,3-oxidosqualene. They are classified into triterpenoid saponins and steroidal saponins, exhibiting diverse bioactivities and applications in the pharmaceuticals, agriculture, cosmetics, and food industries. Beyond their industrial relevance, saponins play a crucial role in plant defense, stress tolerance, and shaping of rhizosphere microbiota. Despite their significant potential, plant-derived triterpenoid saponins remain underexplored. Additionally, their biosynthesis in plants is limited and requires rigorous exploration to decipher novel biotechnological approaches to enhance their production. In this review, we provide a comprehensive update on the types of triterpenoid saponins, the regulation of their biosynthesis pathway, defense responses, therapeutic activities, biotechnological interventions, and the challenges associated with the large-scale production of saponins. Also, we explicitly provide an update on strategies for synthesizing saponins in microbial cells and in vitro plant systems. Thus, this review provides a foundation for further research on plant-derived triterpenoid saponins, their diverse therapeutic activities, their critical role in defense responses, and the development of novel methods to increase saponin production for human health, industrial, and agricultural applications.</p>