The use of hyperspectral imaging to (a) quantify and (b) localise total glucosinolates in florets of a single broccoli species has been examined. Two different spectral regions (vis-NIR and NIR), a number of spectral pre-treatments and different mask development strategies were studied to develop the quantitative models. These models were then applied to freeze-dried slices of broccoli to identify regions within individual florets which were rich in glucosinolates. The procedure demonstrates potential for the quantitative screening and localisation of total glucosinolates in broccoli using the 950-1650 nm wavelength range. These compounds were mainly located in the external part of florets. (C) 2013 Elsevier Ltd. All rights reserved.