A variety of green deep eutectic solvents (DESs) combined with ultrasound-assisted extraction (UAE) were successfully investigated and applied in simultaneous extraction of five bioactive flavonoids in Radix scutellariae. In this paper, 12 DESs were initially screened, then the effective extraction of scutellarin, baicalin, wogonoside, baicalein and wogonin was achieved by using tailor-made DES composed of l-proline and glycerol at a 1:4 molar ratio. Experimental design was performed by response surface methodology (RSM) based on a central composite design (CCD). 50 mg of sample powder was extracted by sonication for 42 min with 1.20 mL of aqueous solution containing 66.7% (v/v) DES, resulting in the optimum extraction efficiency. The recoveries of the five main bioactive flavonoids in Radix scutellariae were in the range of 97.1-100.7% (RSDs were below 2.9%). This established method provided good linearity, precision and accuracy. As a result, the contents of scutellarin, baicalin, wogonoside, baicalein and wogonin in Radix scutellariae (20170910) were 74.05 +/- 1.89 mg g(-1), 176.3 +/- 3.06 mg g(-1), 35.96 +/- 0.38 mg g(-1), 1.76 +/- 0.04 mg g(-1) and 0.60 +/- 0.02 mg g(-1), respectively. The content of baicalin extracted using the Chinese Pharmacopeia (2015) method was 151.3 +/- 2.54 mg g(-1). In comparison using the Student's t-test (p > 0.05), there was no significant difference between the Chinese Pharmacopeia (2015) method and our established method. This work indicates that DESs are promising as green and efficient solvents for analysis of various samples from plant herbs.