Picual olive oils may represent 25% of the world olive oil production. This monovarietal oil is highly valued from a nutritional viewpoint because of its high content of monounsaturated acid and polyphenols. This study reports a method to authenticate virgin olive oils of the Picual variety. The method was based on the very low content of the lignan 1-acetoxypinoresinol in these oils. The compound, as well as the other phenolic substances, was extracted from oil by using N,N-dimethylformamide (DMF), the extract was washed with hexane and nitrogen was bubbled into the solution. The identification and quantification of the lignan used HPLC to separate, followed by UV, electrochemical, fluorescence and mass spectrometry detection or gas chromatography (GC) coupled to a mass spectrometry detector. UV detection offered low sensitivity and peaks coeluting with 1-acetoxypinoresinol were also observed. Mass spectrometry detection clearly discriminated between olive oils of Picual variety and other Spanish varieties. However, lignans show a very high response to fluorescence excitation, this detector is cheaper than mass spectrometry detectors and therefore it was considered as more appropriate for industrial uses. Thus, the latter method allowed discrimination between thirty-eight Picual olive oils and eight oils of the Hojiblanca, Cornicabra, Empeltre and Arbequina varieties. The paper also demonstrated the possibility of analysing all the phenolic compounds in virgin olive oil by fluorescence. Using this technique an easy methodology to authenticate Picual olive oils was developed.