Sesquiterpene lactones (SLs) are one of the most diverse groups of secondary metabolites that mainly have been observed in the Asteraceae. They are composed of a C-15 skeleton bearing functional groups, e.g., hydroxy, keto, or epoxy. Sesquiterpene lactones have been shown to display several biological activities; hence, their therapeutic effects are indispensable. To overcome low yield of sesquiterpene lactone content in native plants, manipulation of their biosynthetic pathway(s) has become an interesting approach for many researchers. Several genetic engineering strategies have been used in plants or microbial systems for elucidation of the biosynthetic pathway and high-level production of sesquiterpene lactones. Here, we will introduce ongoing research and perspectives about the manipulation of sesquiterpene lactone biosynthesis by various non-traditional metabolic engineering strategies, along with successful examples of high-yield production of sesquiterpene lactones mainly focused on parthenolide and artemisinin in plants and microorganisms.