The primary product of the oenological sector is wine. Nonetheless, the grape processing produces large amounts of by-products and wastes, e.g., the grape seeds. In the context of a sustainable production, there is a strong push towards reutilizing these by-products and waste for making useful derivatives since they are rich of bioactive substances with high additional value. As it is true for the wine itself, bringing these by-products derivatives to the market calls for quality measures and analytical tools to assess quality itself. One of the main objectives is to collect analytical data regarding bioactive compounds using potentially green techniques. In the present work, the profile of fatty acids and the main phenolic compounds were investigated by conventional methods. The qualitative analysis of the main functional groups was carried out by Fourier Transform Infrared (FTIR) spectroscopy. Moreover, the successful use of FTIR technique in combination with chemometric data analysis is shown to be a suitable analytical tool for discriminating the grape seeds. Grape seeds of different origin have different content of bioactive substances, making this technique useful when planning to recover a certain substance with specific potential application in health area as food supplement or nutraceutical. For example, Cesanese d'Affile seeds were found to have a rather high fat content with a significant fraction of unsaturated fatty acids. On the other hand, the seeds of Nero d'Avola exhibit the highest amount of phenolic compounds.