In vitro production of secondary metabolites attributed with various medicinal properties has been known since three decades. However, the content of metabolites was found to be very low, limiting their use in meeting the current industrial demands (Kumar et al., 2019). The production of several of these compounds, known as secondary metabolites, is influenced by a wide range of factors such as biotic and abiotic stresses, types of fertilization, agronomic management, elicitors, and, recently, the presence of nanoparticles, without neglecting the use of biostimulators and biocontrollers, in addition to metabolic engineering manipulation. These factors influence both positively and negatively the production of secondary metabolites, giving the plant strategies for its adaptation and survival, as well as compounds with biological activity that contribute to the health of the human being (Garcia-Mier et al., 2019). Information on current in vitro metabolite production strategies, biosynthesis and their applications will open up new doors to design novel genetic interventions, including metabolomics, genomics and tissue culture technologies. Knowledge of in vitro production, the associated challenges and the prospects of attaining high-value bioactive compounds will provide a platform to not only explore but also gain an understanding of the metabolite biosynthesis to enhance phytochemical yields invarious industries (Kumar et al., 2019). In this sense, the present chapter intends to gather relevant and current information on how some of these strategies influence the production of secondary metabolites in plants (Garcia-Mier et al., 2019).