Hops, an essential component in beer, provide flavor, aroma, and microbiological stability. Its chemical composition includes resins, polyphenols, and essential oils, offering anti-inflammatory, antioxidant, and antimicrobial properties. Native to temperate climates, its cultivation has expanded globally, and in the southern hemisphere, hop production is mainly limited by light conditions and day length. However, Chile is a potential contender for hop production due to its agro-climatic diversity. The chemical composition of hops represents a wide variety of compounds, and these are largely determined by the area of cultivation, agronomic factors, drying conditions, and the method of extraction. This study aims to understand the influence of different drying conditions and the supercritical extraction of three ecotypes of Chilean hops to determine the best extraction conditions of antioxidants using response surface methodology (RSM). The effects of drying temperature (50, 60, and 70 °C), extraction pressure with supercritical fluid (150, 200, and 250 bar), and ecotype variety (Ranco, Valdivia and La Unión) on the antioxidant activity were investigated. The Box--Behnken design was employed to optimize parameters in terms of antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, and a Pareto chart was used to identify the most significant factor. The results showed that the optimization of different drying conditions and the supercritical extraction of three ecotypes of Chilean hops were successfully examined using response surface methodology. The quadratic model was well fitted for the response variable, and the measured antioxidant activity in the optimized condition was found in accordance with the predicted value; therefore, it was fitted with the experimental values. We concluded that antioxidant activity highly depends on drying temperature and the extraction pressure with supercritical fluid. Moreover, this is the first report on optimizing antioxidant activity from ecotypes of Chilean hops.