Non-invasive techniques are needed to enable an integrated understanding of plant metabolic responses to environmental stresses. Raman spectroscopy is one such technique, allowing non-destructive chemical characterisation of samples in situ and in vivo and resolving the chemical composition of plant material at scales from microns to metres. Here, we review Raman band assignments of pigments, structural and non-structural carbohydrates, lipids, proteins and secondary metabolites in plant material and consider opportunities this technology raises for studies in vascular plant physiology.