Despite their interests, faba beans are characterised by bitterness but little is known about its compounds that activate the 25 human bitter receptors (TAS2Rs). This study aimed to determine the bitter molecules in faba beans, especially saponins and alkaloids. These molecules were quantified by UHPLC–HRMS in flour, starch and protein fractions of 3 faba bean cultivars. The fractions from the low-alkaloid cultivar and the protein fractions exhibited higher saponin content. Vicine and convicine were highly correlated with bitter perception. The bitterness of soyasaponin βb and alkaloids was studied using a cellular approach. Soyasaponin βb activated 11 TAS2Rs, including TAS2R42 whereas vicine activated only TAS2R16. The high vicine content should explain the faba bean bitterness considering that concentration of soyasaponin βb was low. This research provides a better understanding of the bitter molecules in faba beans. Selection of low-alkaloid ingredients or alkaloid removal treatments could improve the faba bean flavour. Chemical compounds: Soyasaponin βb (PubChem CID: 122097); DDMP soyasaponin (PubChem CID: 74376401); Vicine (PubChem CID: 135413566); Convicine (PubChem CID: 88000)