This work presents a gas chromatography multi-stage mass spectrometry (GC–MS 3 ) method for the determination of ent-kaurene in subcutaneous fat of Iberian pig, present in adipose tissue of animals due to pasture ingestion (extensive fattening system). The method comprises a saponification and a liquid–liquid extraction of the unsaponifiable fraction, followed by an isolation of the hydrocarbon fraction by high performance liquid chromatography (HPLC) and analysis by GC–MS 3 (ion trap) with electronic ionization. The GC–MS 3 analysis allows the isolation and characterization of specific fragments from the original (MS 1 ) molecular structure, and particularly, those fragments originated from the precursor ion (m/z=229) characteristic of ent-kaurene. The MS/MS product fragment m/z=213 is used as a further precursor fragment giving rise to a MS 3 spectrum specific for ent-kaurene. The limit of detection of the MS 3 technique is lower than 0.2μgkg −1 and a linear regression has been found between 0.2 and 112μgkg −1 . This method is applicable for the determination of the fattening system of the Iberian pig.