Background: Flavonoids are components of the daily human diet (fruits and vegetables), and it has been shown to inhibit several kinase enzymes. Due to its kinase inhibitory activity, they are expected to be of great importance in the discovery of new anticancer drugs. Objectives: The objective was to study the cytotoxicity, kinase inhibitory activity, and docking of the isolated aglycones. Materials and Methods: Ultraviolet, high-performance liquid chromatography (LC), nuclear magnetic resonance, and LC-mass spectrometry were used for the identification of the isolated metabolites. Antiproliferative (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and radiometric protein kinase (PK) assays were used to measure the cytotoxicity and PK inhibitory effect of the isolated flavonoids. The docking study on both Aurora B and cyclin-dependent kinase 4 (CDK4)/CycD1 was performed by molecular operating environment (MOE). Results: Luteolin (1), quercetin (2), myricetin (3), apigenin-7-O -β-D-glucopyranoside (4), and kaempferol-7-O -β-D-glucopyranoside (5) were isolated from Scorzonera tortuosissima . Quercetin and myricetin exhibited the highest cytotoxicity against the Michigan Cancer Foundation-7 (MCF-7) (IC50: 5.56 and 7.14 μM, respectively) and against human hepatocellular carcinoma (HepG2) (IC50: 8.61 and 10.31 μM, respectively), while compounds luteolin and apigenin-7-O-β-D-glucopyranoside showed the least cytotoxicity compared to doxorubicin against to MCF-7 and HepG2 (IC50: 2.24 ± 0.85 and 1.82 ± 0.34 μM, respectively). The radiometric PK assay was applied for measurement of kinase inhibitory activity against Aurora B, CDK4/D1, cancer Osaka thyroid, IGF1-R, and FAK kinases, where the aglycone myricetin showed the highest inhibitory activity against Aurora B (IC50: 2.82 μM) and against CDK4/cyclin D1 (IC50: 3.16 μM), while the isolated glycosides 4 and 5 revealed the lowest activity. Docking of the most active compounds 1, 2, and 3 against Aurora B and CDK4/cyclin D1 confirmed its cytotoxic profile. Conclusion: The isolated flavonoids were firstly isolated from S. tortuosissima . The hypothetical mechanism of cytotoxic activity of 1, 2, and 3 on Aurora B and CDK4/cyclin D1 kinases was estimated by in silico study with these enzymes using MOE program.