Qualitative and quantitative profiling of sugars in vegetal materials from Olea europaea cultivars is here reported. Vegetal tissues from olive fruits, leaves, and stems have been characterized by determination of 22 compounds belonging to monosaccharides, disaccharides, trisaccharides, sugar carboxylic acids and alcohols, cyclic polyols, and derived compounds. Sugar isolation was carried out by leaching into a 2:1 dichloromethane/methanol extraction solution under ultrasonic assistance. Multivariate optimization made possible complete isolation of the target fraction in 10 min with an efficiency similar to that provided by a conventional protocol based on 24 h maceration of the vegetal samples. An aliquot of the extract was dried and reconstituted for silylation prior to GC-MS/MS analysis for selective and sensitive identification/quantitation of sugars. Monitoring the target product ions generated after isolation of the precursor ions for each analyte increases the selectivity of the method. The proposed approach is of particular interest for characterization of the sugar fraction in O. europaea, which is of great relevance because of the role of sugars in the metabolism of lipids, proteins, and antioxidants.