Analysis of 50% aqueous methanolic extracts of bark of Quillaja saponaria Molina (quillaja) by liquid chromatography/mass spectrometry (LC/MS), using negative ion electrospray, revealed over 100 saponins. The majority could be assigned to known structures or generalised variations of these from the product ion spectra obtained by serial mass spectrometry in a quadrupole ion trap mass spectrometer. Ten saponins contained a fatty acid domain terminated with both a pentose and deoxyhexose unit, a feature thus far only reported in QS-III. Twenty saponins were based on a hydroxylated derivative of quillaic acid, whereas only six 22β-hydroxyquillaic acid saponins have been described. The occurrence of pairs of saponins differing only by the presence of a rhamnose or xylose unit in the C-3-substituted saccharide was readily observed in two-dimensional mass maps, and these showed the presence of the unreported ‘rhamnose partner’ of QS-III. However, one sample labelled as Q. saponaria appeared to lack all saponins containing rhamnose in the C-3 saccharide. Methods to authenticate saponin extracts of quillaja by LC/MS are suggested based on the general metabolomic profile, the occurrence of specific major saponins covering known structural variations, or the presence of saponins containing the unusual fatty acid domain, revealed by neutral loss analysis. Copyright © 2004 John Wiley & Sons, Ltd.