The investigation presented herein had as its scope the development of an integrated process for the efficient extraction of polyphenols from hop. For this purpose, a novel, natural deep eutectic solvent (DES) was synthesised, composed of glycerol and L-alanine, and the process was optimised by deploying a response surface methodology based on a Box-Behnken design. The variables considered were the DES/water proportion, the liquid-to-solid ratio and the stirring speed. Under the optimised conditions, the yield in total polyphenols achieved was 118.97 ± 8.27 mg gallic acid equivalents per g of dry mass. Ultrasonication, incorporated into the process as a pretreatment step, was shown to significantly change the kinetic pattern of polyphenol extraction and contributed to attaining higher yields only at 80 • C, whereas at lower temperatures a supressing effect was observed. Furthermore, increasing temperature was negatively correlated with the second-order extraction rates, evidencing a slow-down of the extraction rate at elevated temperatures.