At elevated temperatures mineral oil based lubricants are prone to oxidation. Thus, antioxidative stabilization of engine oils is an important issue in lubrication engineering. Although there are various tests in order to assess the oxidative stability of a lubricant, only very little is known about the depletion process of antioxidants on a molecular level. The current study presents a solid-phase extraction method capable of isolating antioxidants and corresponding degradation products from engine oils as well as a high performance liquid chromatography (HPLC) method for their subsequent separation. For detection the HPLC system was coupled to a UV detector and a high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS). These methods not only enable the quantitation of antioxidants employed in lubricants but can also be used for investigations of their corresponding degradation products in used engine oils. By means of HPLC-QTOF-MS and MS/MS experiments it was possible to detect numerous reaction products formed from antioxidants during their service lifetime in engine oils. This allowed a deeper insight into the mode of action of the investigated stabilizers.