Extra virgin olive oil (EVOO) is recognized as one of the healthiest foods for its high content of antioxidants, which forestall and slow down radical formation. Free radical-initiated oxidation is considered one of the main causes of rancidity in fats and oils. As a consequence, reliable protocols for the investigation of oil oxidation based on selective, noninvasive, and fast methods are highly desirable. Here we report an experimental approach based on UV-Vis absorbance, steady-state fluorescence, and electron paramagnetic resonance (EPR) spectroscopy for studying oxidation processes induced by temperature for a period up to 35 d on Sicilian EVOO samples. We followed the decrease in β-carotene content during incubation time and observed changes in polyphenols and tocopherols during the oxidation processes, focusing on the time scale of those changes. Using EPR spectroscopy, the free radical formation in different oil samples is reported, providing a fingerprint for both the antioxidant content and temporal features of the oxidation process at its early stage. PRACTICAL APPLICATION: We monitor β-carotene and chlorophyll in an auto-oxidation process. A protocol based on spectroscopic measurements is presented and can be used for the quality control process of commercial olive oil.