Fatty acids represent major components of cell membranes, serve as energy sources, modulate gene transcription and cell signalling and act as cytokine precursors. It is increasingly apparent that dietary fatty acids influence these vital functions and affect human health. Consequently, analytical techniques are required to identify and quantify the suite of fatty acids present in food and human tissues. Advances in mass spectrometry (MS) offer new opportunities to profile and quantify fatty acids in biological samples. Our aim was to demonstrate the use of GC- atmospheric pressure chemical ionisation (APCI)-ion mobility spectrometry (IMS)-TOF-MS to provide highly specific and sensitive quantification of known fatty acids plus a comprehensive overview of all the eluted analytes. Ionisation was achieved using an APCI source. This new approach was demonstrated on a range of commercial edible oils. Compared to standard GC techniques using flame ionisation detection (FID) or a single quadrupole MS with electron ionisation, GC-APCI-IMS-TOF-MS greatly increased compound selectivity and specificity, leading to greatly enhanced confidence in fatty acid methyl esters (FAME) identification and quantification. Our approach also added the fingerprint of high-resolution accurate mass (HRAM) discovery data, with collision cross section (CCS) values, relating to many other analytes. This method can be readily applied to study food provenance, food fraud and to identify fatty acid related illnesses.