Lentil saponins are triterpene glycosides, mainly soyasaponins I and betag (also known as VIota), with multiple health-promoting properties. This paper reports the isolation of soyasaponins I and betag from soybeans as analytical standards and the development of a new analytical procedure for quantification of their content in various cultivars of Italian lentils, by SPE-HPLC-MS. Soyasaponins I and betag were isolated from soybeans at a purity of >90% and characterized by MS/MS (ion trap) experiments. The determination of soyasaponins in lentils was performed by extraction, SPE purification, and HPLC-MS (single quadrupole) analysis; results were confirmed by MALDI-TOF experiments. Calibration curves for soyasaponin I and betag showed correlation coefficients of 0.998 and 0.997, respectively. LOD and LOQ values were 0.02 and 0.2 mg kg(-1) for soyasaponins I and 0.1 and 1 mg kg(-1) for soyasaponin betag. Recoveries calculated at a 100 mg kg(-1) fortification level ranged from 85 to 97%, with n = 10 and RSDs of <12%. In the 32 lentil samples, contents of soyasaponin I ranged from 28 to 407 mg kg(-1), whereas that of soyasaponin betag ranged from 110 to 1242 mg kg(-1).