Publisher Summary Saponins constitute an important class of secondary metabolites from plant and animal domains. They display a broad spectrum of biological activities that have raised the interest of phytochemists for the past 40 years. With molecular weights ranging from 600 to 2000 daltons, their structures are complex. This chapter describes the current techniques in use for the structural elucidation of saponins. From a chemical standpoint, saponins are made of three entities: an aglycone (steroid or triterpene), sugars, and sometimes acids. The determination of the structure therefore requires identification of the elements and sequencing. The elements of a saponin may be identified either on the intact saponin or after hydrolytic cleavage. It is always better to perform the identification on the intact saponin to eliminate the possibility of artefact formation during hydrolysis. This identification can be realized by mass spectrometry (MS) or nuclear magnetic resonance. Artefacts are most often formed when the aglycone is sensitive to acid.