A sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI‐MS/MS) method was developed for the identification and quantification of two antihepatotoxic coumarinolignoids cleomiscosin A and cleomiscosin B in different extracts of the seeds of Cleome viscosa. The separation of cleomiscosin A and cleomiscosin B was achieved on an RP₁₈ column using a solvent system consisting of a mixture of acetonitrile–methanol (1:2, v/v) and acetonitrile–water–formic acid (5:95:0.3, v/v) as a mobile phase in a gradient elution mode. A multiple‐reaction monitoring (MRM) method was developed for quantification of cleomiscosin A and cleomiscosin B in the seed extracts of Cleome viscosa. On the basis of signal‐to‐noise ratio of 3, the limit of detection in MRM mode for cleomiscosin A and cleomiscosin B were 1.0 and 4.0 ng/mL respectively. The method was validated in terms of linearity, accuracy and precision for 6 days. The method developed was found to be useful for identification and quantification of cleomiscosin A and cleomiscosin B in the different extracts of the seeds of Cleome viscosa. Copyright © 2008 John Wiley & Sons, Ltd.