Kale is a member of the Brassicaceae family and has a complex profile of flavonoid glycosides. Therefore, kale is a suitable matrix to discuss in a comprehensive study the different fragmentation patterns of flavonoid glycosides. The wide variety of glycosylation and acylation patterns determines the health-promoting effects of these glycosides. The aim of this study is to investigate the naturally occurring flavonoids in kale. A total of 71 flavonoid glycosides of quercetin, kaempferol and isorhamnetin were identified using a high-performance liquid chromatography diode-array detection/electrospray ionization multi-stage mass spectrometry (HPLC-DAD/ESI-MS(n)) method. Of these 71 flavonol glycosides, 27 were non-acylated, 30 were monoacylated and 14 were diacylated. Non-acylated flavonol glycosides were present as mono-, di-, tri- and tetraglycosides. This is the first time that the occurrence of four different fragmentation patterns of non-acylated flavonol triglycosides has been reported in one matrix simultaneously. In addition, 44 flavonol glycosides were acylated with p-coumaric, caffeic, ferulic, hydroxyferulic or sinapic acid. While monoacylated glycosides existed as di-, tri- and tetraglycosides, diacylated glycosides occurred as tetra- and pentaglycosides. To the best of our knowledge, 28 compounds in kale are reported here for the first time. These include three acylated isorhamnetin glycosides (isorhamnetin-3-O-sinapoyl-sophoroside-7-O-D-glucoside, isorhamnetin-3-O-feruloyl-sophoroside-7-O-diglucoside and isorhamnetin-3-O-disinapoyl-triglucoside-7-O-diglucoside) and seven non-acylated isorhamnetin glycosides.