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Celastrus hindsii is a potential source of flavonoids with biological activities. This study aimed to develop an ultrasound-assisted technique for extracting flavonoids from leaves of C. hindsii . Response surface methodology was employed to optimize the extraction conditions for maximizing the total flavonoid content (TFC). A maximum TFC of 23.6 mg QE/g was obtained under the extraction conditions of ultrasonic power of 130 W, extraction temperature of 40°C, extraction time of 29 min, and ethanol concentration of 65%. The flavonoid-rich extracts were then studied for their antioxidant and anticancer activities. The results showed that the C. hindsii leaf extract exhibited potent radical scavenging activities against DPPH (IC 50 of 164.85  μ g/mL) and ABTS (IC 50 of 89.05  μ g/mL). The extract also significantly inhibited the growth of 3 cancer cell lines MCF7, A549, and HeLa with the IC 50 values of 88.1  μ g/mL, 120.4  μ g/mL, and 118.4  μ g/mL, respectively. Notably, the extract had no cytotoxicity effect on HK2 normal kidney cell line. This study suggests that flavonoid-rich extract is a promising antioxidant and anticancer agent and that ultrasound-assisted extraction is an efficient method for extracting flavonoids from C. hindsii leaves., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2020 Dinh-Chuong Pham et al.)