Two minor triterpene saponins, one previously not reported, were isolated from the underground parts of Lysimachia thyrsiflora L. The structures were determined based on a combination of one- and two-dimensional NMR techniques, and mass spectrometry, as 30- O-β-D-glucopyranosyl-3β,16α,30-trihydroxy-olean-12-en-28-yl acetate 3- O-β-D-glucopyranosyl-(1→4)- O-[β-D-glucopyranosyl-(1→2)-]- O-α-L-arabinopyranoside (denoted as thyrsiloside A), and davuricoside L. Quantitative determination of these two compounds, along with other saponins present in L. thyrsiflora underground parts, was performed by a developed UPLC-ESI-MS/MS method, validated according to the ICH guidelines. The relative total content of triterpene saponins amounted to 1.19 μg/mg dw. Both 4 and 5 were present in minor quantities, 0.031 and 0.077 μg/mg dw, respectively. Assays performed on a panel of human cancer cell lines showed antiproliferative activity against prostate carcinoma PC-3 (GI50 after 48 h = 20 and 24 μg/mL, respectively).