Faba beans exhibit environmental, agronomic, food functional, and nutritional benefits, and are a promising alternative to animal proteins. The presence of off-flavours, and in particular bitterness, is a barrier to their consumption. However, little is known about the compounds of pulses that activate the 25 human bitter taste receptors (TAS2Rs). To better understand the role of the saponins and alkaloids in faba bean bitterness, three cultivars and three fractions (flour, starch and protein) obtained by air-classification were studied for their bitter properties. First, the identification and quantification of these molecules was carried out using UHPLC–HRMS (ultra-high-performance liquid chromatography–high-resolution mass spectrometry). The low-alkaloid cultivar fractions and the protein fractions exhibited higher saponin content. Second, these data were correlated with the bitter intensity of the fractions evaluated by a trained panel. Only the two alkaloids, vicine and convicine, were highly correlated with the bitter perception. Third, the bitterness of the two alkaloids and soyasaponin βb was studied using a cellular-based functional taste receptor assay. Vicine only activated TAS2R16 whereas 11 TAS2Rs were activated by soyasaponin βb. However, due to the low concentration of soyasaponin βb and the high concentration of vicine, the origin of the faba bean bitterness is highly suspected to be related to vicine. This research leads to a better knowledge of the compounds involved in the bitterness of faba beans and the effect of cultivar and air-classification on their bitter content. Choosing ingredients with low-alkaloid content or using alkaloid removal treatments could improve the flavour of faba beans and increase the consumer acceptability.