4‐Hydroxy‐2(E)‐nonenal (HNE), a product of lipid peroxidation, has been extensively studied in several areas,including metabolism with radio‐isotopes and quantification in various matrices with deuterium‐labelled HNE asstandard. The aim of this work was to evaluate the relevance of 13C‐labelled HNE in biotransformation studiesto discriminate metabolites from endogens by liquid chromatography/electrospray ionisation mass spectrometry(LC/ESI‐MS). 13C‐LabelledHNEwas synthesised in improved overall yield (20%), with the incorporation of two labelsin the molecule. Immortalised mouse colon epithelial cells were incubated with 2:3 molar amounts of HNE/13C‐HNEinorder to gain information on the detection of metabolites in complex media. Our results demonstrated that the stableisotope m/z values determined by mass spectrometry were relevant in distinguishing metabolites from endogens, andthat metabolite structures could be deduced. Six conjugate metabolites and 4‐hydroxy‐2(E)‐nonenoic acid wereidentified, together with an incompletely identified metabolite. Stable‐isotope‐labelled HNE has already been usedfor quantification purposes. However, this is the first report on the use of 13C‐labelled HNE as a tracer for in vitrometabolism. 13C‐Labelled HNE could also be of benefit for in vivo studies.