Steroidal glycoalkaloids (SGAs) extracted from tomato leaves and berries (Lycopersicon esculentum Mill.) were separated and identified using optimized reversed-phase liquid chromatography with electrospray ionization (ESI) and ion trap mass spectrometry (ITMS). The ESI source polarity and chromatographic conditions were evaluated. The ESI spectra contain valuable information, which includes the mass of SGAs, the mass of the aglycones, and several characteristic fragment ions. Cleavage at the interglycosidic bonds proximal to the aglycones is the most prominent process in the ESI process. A protonated molecule, [M+H]+, accompanied by a mixed adduct ion, [M+H+Na]2+, was observed for alpha-tomatine (i.e., m/z 1034.7 and 528.9) and dehydrotomatine (i.e., m/z 1032.6 and 527.9) in positive ion mode spectra. The structures of these tomato glycoalkaloids were confirmed using tandem mass spectrometry. The identification of a new alpha-tomatine isomer glycoalkaloid, named filotomatine (MW 1033), which shares a common tetrasaccharide structure (i.e., lycotretraose) with alpha-tomatine and dehydrotomatine, and soladulcidine as an aglycone, is described for the first time. It occurs in significant amounts in the extracts of wild tomato foliage. Multistage mass spectrometry both of the protonated molecules and of the doubly charged ions was used for detailed structural elucidation of SGAs. Key fragmentations and regularities in fragmentation pathways are described and the fragmentation mechanisms involved are proposed.