Abstract The potential of protein fractionation hyphenated to mass spectrometry (MS) to detect and characterize the transgenic protein present in Roundup Ready™ soya and maize has been investigated. Genetically modified (GM) soya and maize contain the 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene from Agrobacterium tumefaciens CP4 , which confers resistance to the herbicide glyphosate. The GM soya and maize proteomes were fractionated by gel filtration, anion‐exchange chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‐PAGE) prior to MS. This facilitated detection of a tryptic peptide map of CP4 EPSPS by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS and nanoelectrospray ionization quadrupole time‐of‐flight (nanoESI‐QTOF) MS. Subsequently, sequence information from the CP4 EPSPS tryptic peptides was obtained by nanoESI‐QTOF MS/MS. The identification was accomplished in 0.9% GM soya seeds, which is the current EU threshold for food‐labeling requirements. Copyright © 2007 John Wiley & Sons, Ltd.