The determination of sterols and fatty alcohols is a part of the study of the metabolomic profile of the unsaponifiable fraction in olive tree. Leaves and drupes from three varieties of olive tree (Arbequina, Picual, and Manzanilla) were used. The content of the target compounds was studied in five ripeness stages and three harvesting periods for olive drupes and leaves, respectively. A method based on ultrasound-assisted extraction and derivatization for the individual identification and quantitation of sterols and fatty alcohols, involving chromatographic separation and mass spectrometry detection by selected ion monitoring, was used. The concentrations of alcohols and sterols in the drupes ranged between 0.1 and 1086.9 mug/g and between 0.1 and 5855.3 mug/g, respectively, which are higher than in leaves. Statistical studies were developed to show the relationship between the concentration of the target analytes and variety, ripeness stage, and harvesting period.