Fluorescence spectroscopy is a reliable and fast method for determining the deterioration of extra virgin olive oil (EVOO). EVOO was analysed by HPLC for determining antioxidant changes in EVOO due to heating at 170°C for 3 h. This thermal oxidation caused a significant exponential decrease in hydroxytyrosol (OH-Tyr) and vitamin E (vitE), while the reduction in the tyrosol content was relatively small. Hydroperoxydes were analysed by an indirect colorimetric method. Their content decreased exponentially during the heating process. Fluorescence excitation spectra with emission wavelength at 330 nm were recorded to monitor the evolution of polyphenols and vitE, while fluorescence excitation spectra with emission at 450 nm were used to monitor the evolution of hydroperoxydes. Results of partial least-square calibration models (PLS) show that the degradation of polyphenols, vitE and hydroperoxide, as well as formation of fluorescent components derivatives can be quantified with fluorescence spectroscopy. Application of chemometrical data analysis allows to optimise the extraction of the information contained in the data. Fluorescence can therefore be used as a rapid technique for evaluating the quality of heat-treated EVOO.