Triterpenoids are secondary metabolites synthesized by oxidosqualene cyclases (OSCs), a class of enzyme thatconverts the linear squalene-2,3-oxide to single or multi-ring products in a dynamic process called cyclization.The cyclization cascade is powered by a migrating carbocation that through a series of 1,2-methyl and hydrideshifts which gives a series of conformationally discrete carbocation intermediates, with the loss of a protongenerating a neutral reaction product. Seco-triterpenoids are a type of triterpenoids with an opened ring whichmay derive either from an unusual deprotonation process of an OSC or from post cyclization modificationsteps. The bioactivity and biosynthesis of the seco-triterpenoids is intriguing, and yet underexplored.In this thesis a review of structure-activity relationships of seco-triterpenoids is presented. Seco-cycloartanesreport the highest cytotoxicity, while seco-dammaranes displayed anti-retroviral activities and 3,4-secooleananeshad neuroprotective activities. In addition, the distribution of seco-triterpenoids across the plantphylogeny reveals they are restricted to specific plant families. Finally, the occurrence of similar secotriterpenoidsin distantly related plant families hints that seco-triterpenoid pathways may result fromconvergent evolution.The hypothesis that seco-triterpenoids pathways evolve convergently was tested by studying the α-onocerinpathway in Ononis spinosa and Lycopodium clavatum. These led to the characterization of two OSCs in O.spinosa (OsONS1 and OsONS2) which both are able to produce α-onocerin. Contrary to the α-onocerin pathwayin L. clavatum in which two different cyclases are needed for α-onocerin synthesis. Moreover, an OSCphylogenetic analysis indicated the onocerin synthases of O. spinosa arose from a different evolutionary routethan those in L. clavatum. In addition, two squalene epoxidases (SQEs) of O. spinosa specialized for onocerinproduction were described and named OsSQE1 and OsSQE2. These SQEs displayed protein-protein interactionswith OsONS1 and proved to boost the onocerin production of OsONS1. Finally, protein-protein interactionsbetween enzymes from the α-onocerin and cucurbitacin pathways were tested and the results revealed thatprotein-protein interactions are not conserved amongst SQEs and OSCs from different plant species.As a partner of the EU-funded project TriForC, our lab was subject to screen several plant species for bioactivetriterpenoids. Thus, extracts from 29 plant species were prepared and 87 fractions were obtained by solidphase extraction. Bioactivity screening of these fractions was performed by other TriforC partners.With this thesis, we have paved the way for exploring pharmaceutical valuable seco-triterpenoids, and havealso provided novel insight into triterpenoid biosynthesis and evolution. This knowledge will increase theopportunity for sustainable production and commercial utilization of seco-triterpenoids.