Phenomic approaches have made enormous strides in mapping and modelling plant growth. Applying similar noninvasive methods to investigate metabolites in planta is still developing. In this paper I compare a range of spectroscopic methods to investigate the presence, concentration and localization of secondary metabolites, water and protein in leaves of trees and crops such as sorghum. One of the big challenges is detecting and interpreting the chemical signature against the background of water. For example, cyanogenic glucosides, anti-herbivore defense compounds found in over 3000 species, have a distinctive chemical signature arising from the triple nitrile bond but this is often obscured by other metabolites and especially the signature from water. Here I compare the use of Fourier-Transform Infrared focal plane array imaging, Near Infra-Red Spectroscopy, Raman microspectroscopy and MALDI-MS imaging to map the in situ localization of secondary metabolites in sorghum coleoptiles and seeds. Water has weak Raman scattering properties, making it possible to determine in situ localization. Varieties of sorghum that lack cyanogenic glucosides, selected from our EMS-TILLING population, were used as a control. These visualization techniques give novel insights into the link between cyanogenic glucoside production and potential roles in resistance to environmental stress.