Sulforaphane (1-isothiocyanato-4-(methylsulfinyl)-butane) and iberin (1-isothiocyanato-3-methylsulfinylpropane) have attracted greatest attention due to their anti-inflammatory and cancer-preventive properties. These isothiocyanates are products of the enzymatic hydrolysis of the glucosinolates glucoraphanin and glucoiberin, contained only in the plants of the order Brassicales. Cruciferous vegetables such as broccoli, cabbage and cauliflower, belong in the order Brassicales and specifically in the Brassicaceae family. Our aim was to develop an efficient and accurate method for the simultaneous determination of sulforaphane and iberin in cruciferous vegetables using Ultra-high Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-ToF-MS). The method was applied for the quantitative determination of these compounds in a variety of cruciferous vegetables (green and purple broccoli, white and purple cabbage, radish, turnip, arugula, watercress, and cauliflower). The results showed that green and purple broccoli contained the highest levels of sulforaphane (660.14±34.29 to 210.11±9.76 μg g^-1 dry weight) while the highest concentration of iberin was detected in purple broccoli (144.98±3.56 μg g^-1 dry weight). The lowest concentrations of sulforaphane and iberin were measured in watercress and radish. The differences in the content of these compounds can be attributed to the variability among Brassicaceae species, geography, season, and various environmental factors.