Ginsenosides containing different numbers of glycosyl groups can be easily differentiated based on the formation of characteristic ginsenoside-acetate adduct anions and deprotonated ginsenosides generated by electrospray ionization (ESI) of methanolic solutions of ginsenosides (M) and ammonium acetate (NH4OAc). Ginsenosides containing two glycosyl groups gave a characteristic mass spectral pattern consisting of [M+2OAc]2−, [M−H+OAc]2− and [M−2H]2− ions with m/z values differing by 30 Th, while this mass spectral pattern was not observed for ginsenosides containing one glycosyl group. Formation of [M+2OAc]2− was influenced by the chain length of glycosyl groups and was used to differentiate the ginsenosides containing different combinations of monosaccharide and disaccharide units in the glycosyl groups. Under identical collisional activation conditions, [M+OAc]−, [M−H+OAc]2− and [M+2OAc]2− underwent proton abstractions predominantly to generate [M−H]−, [M−2H]2− and [M−H+OAc]2− ions, respectively. The ion intensity ratios, I/I, I/I and I/I, being sensitive to the structural differences of ginsenosides, could differentiate the isomeric ginsenosides, including (i) Rf, F11 and Rg1, (ii) Rd and Re, and (iii) Rb2 and Rc. Additionally, NH4OAc was found to enhance the sensitivity of detection of ginsenosides in the form of [M−H]− down to the femtomole level. Copyright © 2006 John Wiley & Sons, Ltd.