To reveal varietally differing glucosinolate (GSL) contents in radish (Raphanus sativus L.) cultivated in Japan, the total and individual GSLs of 28 cultivars were analyzed using high-performance liquid chromatography. In these cultivars, GSL types including three aliphatic GSLs (glucoraphenin, glucoerucin, and 4-methylthio-3-butenyl GSL (4MTB-GSL)) and three indolyl GSLs (4-hydroxy-glucobrassicin, glucobrassicin, and 4-methoxy-glucobrassicin) were detected. No cultivar-specific type of GSL was identified. The dominant GSL was 4MTB-GSL, but its contents differed remarkably: 8.6 μmol/g in ‘Koushin’ to 135.7 μmol/g in ‘Karami 199’. Over about 90% of all GSLs in Japanese radish type are 4MTB-GSL, a higher percentage than in Chinese or European garden radish cultivars. A simple, rapid method for estimating total GSL contents in crude extracts was established because of the small variation of glucosinolate composition in Japanese cultivars. The total GSL content can be estimated using an equation for prediction with absorbance at 425 nm in a mixture of GSL crude extract and palladium (II) chloride solution: Total GSL (μmol/g) = 305.47 × A₄₂₅ − 29.66. Its coefficient of determination (R²) and standard error of prediction (SEP) are 0.968 and 8.052. This method enables total GSL content estimation from more than 200 samples per person per day.