High-resolution electrospray ionization multistage tandem mass spectrometry (MS 1-7) in negative ion mode was used to determine the accurate masses and fragmentation pathways of two compounds, 4'-demethylepipodophyllotoxin and 4'-demethyl-4-azido-4-deoxyepipodophyllotoxin, which are key intermediate compounds for the preparation of podophyllotoxin-type anti-cancer drugs. The deprotonated molecules [M-H]* of both compounds were readily observed in the conventional single-stage mass spectra due to the presence of the phenolic hydroxyl group in the molecules. Abundant information on the product ions was obtained from tandem mass spectra (MS 2-7) in negative ion mode. Based on the exact masses acquired from 14 different tandem mass spectra, a similar MSn fragmentation pathway was proposed for both compounds. A characteristic product ion produced in the MS 2-4 product ion scan experiments is the cyclohexylenetrione anion [M-H-2Me-RH]* or [M-H-RH-2Me]* at m/z 351 (C19H11O7) formed by the consecutive losses of two CH3 radicals at the 3'- and 5'-positions and the neutral loss of RH, where R = a 4-substituted group (-OH or -N3), from the [M-H]* ion. This anion may be considered as diagnostic for the presence of this type of compound. The other common cleavages are the neutral losses of CO at least two times in the MS 6,7 product ion spectra. The results of this work could serve as an effective tool for the detection or determination of other derivatives of 4'-demethyl-4beta-substituted podophyllotoxin, which are widely used as intermediates for the preparation of anti-tumor drugs.