In the current study, Hibiscus sabdariffa has been extracted by microwave-assisted extraction (MAEX). Citric acid-based deep eutectic solvents (DES) have been specially designed, where a hydrogen bond donor (HBD) (glycerol and ethylene glycol) and a hydrogen bond acceptor (HBA) (citric acid) with a certain molar ratio (1/4) were used. After the best DES (citric acid/ethylene glycol) has been decided to extract the bioactive ingredients, operation conditions (power of microwave, volume of solvent water and content in the DES) of the MAEX for the relevant plant material have been optimized through Box-Behnken design (BBD) of response surface approach (RSA). The maximum yields of total phenolics (TP), total anthocyanins (TAA) and antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) have been found as 31.897 mg-GAE/g-DH, 2.961 mg-C3G/g-DH and 95.887% under the optimal conditions (35 mL DES including 50% water (v/v) at 550 W power of microwave). The differences between the experimental and estimated findings were lower than 2%. Three replicate test results obtained by in vitro experiments were also statistically analysed by using one-way analysis of variance (ANOVA) test.