A simple and rapid method for the extraction of crude glucosinolates from powder of freeze-dried radish roots was developed using a common solvent, methanol/water = 80: 20 (v/v) at room temperature. Hot methanol is not required for the extraction procedure of this new method. Both 4-methylthio-3-butenyl glucosinolate, which is a major glucosinolate in radish root, and total glucosinolates of Japanese radish cv. ‘Karami 199’ were quantified using HPLC. The amounts of glucosinolate extracted using this new method were 4–6% higher than those obtained using the conventional method with hot methanol. To compare the new method with the conventional method, the amounts of glucosinolate extracted from roots of eight cultivars in Japanese radish and five breeding lines were estimated. A high positive correlation was found between them. This simple and rapid method 1) obviates hot methanol for glucosinolate extraction and 2) requires only one-third of the extraction time of the conventional method, enabling glucosinolate extraction from about 150 samples by one person in a single day.