Hyperforin is a major active constituent ofHypericum perforatum(St. John’s wort). It has amazing phar-macological activities, such as antidepressant properties, but it is labile and difficult to synthesize. Its sen-sitivity and lipophilicity are challenges for processing and formulation. Its chemical complexity provokes approaches of biotechnological production and modification. DedifferentiatedH. perforatumcell cultures lack appropriate storage sites and hence appreciable hyperforin levels. Shoot cultures are capable of forming hyperforin but less suitable for biomass up-scaling in bioreactors. Roots commonly lack hyper-forin but a recently established adventitious root line has been demonstrated to produce hyperforin and derivatives at promising levels. The roots also contained lupulones, the typical constituents of hop (Humulus lupulus). Although shear-sensitive, these root cultures provide a potential production platform for both individual compounds and extracts with novel combinations of constituents and pharmacolog-ical activities. Besides in vitro cultivation techniques, the reconstruction of hyperforin biosynthesis in microorganisms is a promising alternative for biotechnological production. The biosynthetic pathway is under study, with omics-technologies being increasingly implemented. These biotechnological approaches may not only yield hyperforin at reasonable productivity but also allow for modifications of its chemical structure and pharmacological profile.