High-resolution electrospray ionization multistage tandem mass spectrometry (MS1–9) was used to determine the accurate masses and the fragmentation pathways of protonated podophyllotoxin (1) and its corresponding 4′-demethyl-4β-substituted derivatives (2–4). The protonated molecules, [M + H]+, of all the four compounds were observed in the conventional single-stage mass spectra. Two fragmentation pathways, that appear to be characteristic of the four compounds, are proposed on the basis of their multistage tandem mass spectrometric data. The characteristic elimination, from the precursor protonated ions, of the neutral groups 4-R1H, 1-ArH, CO, CH2O and C4H4O2, in which R is located on C-4, is the common elimination, and the product ions at m/z 267, 239, 229, 181, 173, 153, 143 and 115 are the common diagnostic masses. The elimination of the R1 group substituent located on the C-4 position of compounds 1–4 has a significant influence on the fragmentation pathway obtained in the conventional single-stage mass spectra. A large R1 group would be unfavorable for this elimination, unless the collision energy is raised. Apart from the common fragmentations obtained for the protonated molecules 1–4, significant additional product ions were detected in the various multistage tandem mass spectrometric analyses, particularly in the case of the product ions derived initially from the phenolic hydroxyl group of 2–4, which are different from those of 1. Based on these additional formed product ions, several additional fragmentation pathways for 1 or 2–4 are also presented. Copyright © 2007 John Wiley & Sons, Ltd.