In recent years, there has been increasing interest in the use of red beets as a valuable source of bioactive compounds. Although saponins may be responsible for the pharmacological and nutraceutical activities of red beets, information on the saponin composition of various red beet cultivars is very limited, and the saponin profiles remain to be fully elucidated. Here, we profiled triterpene saponins from the flesh and skin of different red beetroot cultivars, whose names of Polish cv. are Bonel, Chrobry and Nochowski. In addition, the total triterpene saponin contents in cultivars Bonel, Chrobry, Nochowski and in their various root parts were determined. Triterpene saponins isolated from the skin and flesh of different red beetroot cultivars were qualitatively profiled using two high-performance liquid chromatography systems (HPLC-ESI-IT MS and UPLC-HESI-Q-Orbitrap MS). The total triterpene saponin contents in different cultivars were analyzed using high-performance liquid chromatography-tandem mass spectrometry. In total, 40 triterpene saponins were tentatively identified, and among them, 5 compounds were potentially new saponins, not previously reported in red beets. These saponins are new to red beets and were reported in B. vulgaris for the first time. The analyzed cultivars were different in terms of their total saponin content. The results of our study indicated that the variation in the composition of saponins depended on the red beet cultivars and part of the roots. Higher concentrations of these compounds were found in the skins than in the flesh of roots. We developed an analytical strategy to discover and identify triterpene saponins from B. vulgaris L. using a combination of techniques to detect the comprehensive fragmentation pathways of the saponins present at low concentrations in plant material.