Glucosinolates and their enzyme-degraded metabolites, such as isothiocyanates, have shown great potential to prevent and treat chronic-degenerative diseases. Broccoli sprouts (Brassica oleracea L., var. italica) are an excellent source of glucosinolates. The objective of this study was to evaluate different process conditions (temperature, solvent concentration, and sample:solvent ratio) for the extraction of glucosinolates present in broccoli sprouts. The study evaluated different combinations of temperature (15–64 °C), ethanol concentration (0–100%), and sample:solvent ratio (1:15–1:35 w/v). The treatment with 40 °C, 50% ethanol/water (v/v), 1:35 (w/v) generated the highest extraction of total glucosinolates (100,094 ± 9016 mg/kg DW). The ethanol percentage was the factor that exerted the greatest impact on glucosinolate extraction (p < 0.05). In addition, the amount of glucoraphanin in the extract was determined, obtaining a final concentration of 14,986 ± 1862 mg/kg DW of sprouts processed under the optimal extraction conditions studied. The data presented herein demonstrate the relevance of the ethanol concentration during the extraction and the importance of applying high temperature in solid-liquid phase extraction. Under optimal conditions, it was possible to obtain extracts rich in glucosinolates to prepare food supplements, nutraceuticals, and even pharmaceuticals with application in the prevention and treatment of chronic-degenerative diseases.