During the harvesting of the broccoli plant, the leaves are discarded, being considered a by-product that may be up to 47% of total broccoli biomass, representing a large amount of wasted material. The use of broccoli leaves is of great interest in the sense that this wasted material is rich in health promoter compounds, such as isothiocyanates. In this study, C57BL/6J mice ingested 790 mg/kg broccoli leaf flour, and the presence of glucosinolates and isothiocyanates in the plasma, liver, kidney, adipose tissue, faeces and urine was analysed at 1, 2, 4, 8, 12 and 24 h post-ingestion. In plasma, only glucoerucin (GE), glucobrassicin (GB), sulforaphane (SFN) and indol-3-carbinol (I3C) were detected, and all four compounds peaked between 4 and 8 h after ingestion. The compounds SFN, SFN-glutathione (SFN-GSH), SFN–cysteine (SFN-CYS) and SFN-N-acetyl-cysteine (SFN-NAC) were excreted in faeces at high levels, while glucoraphanin (GR), the precursor of SFN, was not detected in any biological samples other than urine. In the liver, the compounds GE, SFN-CYS, SFN-NAC and I3C were detected, while in the kidney, only GE, GB and SFN-GSH were present. None of the glucosinolates and isothiocyanates analysed were detected in fat tissue. These results demonstrate that glucosinolates and their derivatives were absorbed into the bloodstream and were bioavailable after ingestion of powdered broccoli leaves.