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“Suspect screening metabolomics” is a mid-way approach between “targeted” and “untargeted” analysis. For this aim, a new database of putative grape and wine metabolites (GrapeMetabolomics ) was expressly constructed. Currently, this database contains around 1,100 compounds. By performing UHPLC/QTOF mass spectrometry analysis in both positive and negative ionization mode, in a grape extract averaging 320–450 putative compounds are identified. Most of these compounds are important grape metabolites, including flavonols, anthocyanins, and stilbene derivatives. In the present study, this approach was focalized on the characterization of flavonols of 18 important Italian red and white grape varieties and the method provided the identification of 15 flavonols. By performing statistical analysis (Principal Component Analysis and Cluster Analysis), the effect of the variety on the flavonol composition of the grapes was studied. Both the red and white samples fell into three different groups, respectively, on the basis of their flavonol profiles. Because the samples were cultivated in the same vineyard, their profile potentially was not affected by cultural or environmental factors. Anyway, these preliminary results will have to be confirmed by the study of grape samples collected in different years and from different vineyards.