Abstract Seven ecdysteroids isolated from Sida rhombifolia L. were studied by electrospray ionization multi‐stage tandem mass spectrometry (ESI‐MS n ) in the positive ion mode using an ion trap analyzer and high‐performance liquid chromatography coupled with a diode‐array detector (HPLC/DAD). The HPLC experiments were performed by means of a reversed‐phase C 18 column and a binary mobile phase system consisting of water (containing 0.05% formic acid) and acetonitrile (containing 0.05% formic acid) under gradient elution conditions. According to mass spectral features and the substitution at C‐2, C‐20, C‐24 and C‐25, ecdysteroids in S . rhombifolia were classified into three sub‐groups. Structural identification of these three sub‐groups of ecdysteroids was established by LC/multi‐stage ion trap mass spectrometry on‐line or off‐line. The fragmentation patterns of ecdysteroids yielded ions of successive loss of 1–4 water molecules. Furthermore, ions corresponding to the complete loss of the side chain at C‐17 will help to identify the sub‐groups of ecdysteroids in addition to containing a hydroxyl moiety at one of the above‐mentioned positions. Based on the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by ESI‐MS n spectra, a total of nine naturally occurring ecdysteroids were identified, of these two are identified for the first time in S. rhombifolia . Copyright © 2008 John Wiley & Sons, Ltd.