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Mulberry leaves have been widely used to produce various health products. In this paper, an efficient procedure of ultrasound-assisted hydroalcohol-acid extraction (UAHAE) was established to estimate the total flavonoids content in mulberry leaves by quantifying their resulting aglycones (quercetin and kaempferol) using HPLC-DAD. Effective hydrolysis of glycosides to aglycones was achieved in an ethanol-HCl-water (7/2/1, v/v/v) solution at 75°C by ultrasound (40 kHz) for 60 min. The average contents of quercetin and kaempferol were 7.12 mg/g and 2.13 mg/g, respectively, in the variety of cultivar MC308, and their recoveries were 105.48% and 105.81%, respectively. The total flavonoid content of most varieties was between 4-10 mg/g, accounting for 80% in 86 species of mulberry leaves. With the increase in leaf maturity, the general trend of the change in flavonoid content was a decrease at first and then an increase. The highest total flavonoid content in commercial mulberry tea (CT 2) is 4.76 mg/g, and the DPPH radical scavenging activities were similar to the distribution of total flavonoids measured by UAHAE in three types of mulberry tea. These finding provide a basis for industrial-scale manufacturing of edible medicinal mulberry leaf products.