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The production and isolation of secondary metabolites through in vitro plant culture techniques represent a sustainable and versatile alternative to traditional cultivation. This review provides an integrative overview of the principles, methods, and advances in in vitro systems—such as callus and suspension cell cultures, hairy root cultures, micropropagation, somatic embryogenesis, and protoplast culture—highlighting their ability to exploit plant cell totipotency for the controlled synthesis of bioactive compounds with pharmaceutical, nutraceutical, and cosmetic value. These systems enable scalable, reproducible, and environmentally responsible production, supported by innovations in elicitation, metabolic engineering, and omics technologies that enhance yield and stability. Despite challenges such as high establishment costs and species-specific requirements, in vitro approaches ensure continuous metabolite production independent of climatic or ecological factors. Moreover, these biotechnological strategies open new opportunities to explore and preserve global plant biodiversity—particularly in regions rich in endemic flora, such as Ecuador—while promoting equitable access to scientific and industrial applications. Ultimately, in vitro plant culture serves as both a platform for secondary metabolite discovery and a pathway toward sustainable, inclusive, and socially impactful biotechnological development.