An enzymatic assay method in 96-well microplates (MP method) is proposed for non-structural carbohydrates (NSC) in ligneous plants. Soluble sugars were extracted from 50 mg of finely ground plant powder, in the presence of water, methanol and chloroform. The glucose, fructose and sucrose contents were successively determined in each well, although sorbitol could not be assayed under these conditions. Whatever the sugar, the precision (reproducibility), linearity (addition of specific amounts of sugars) and accuracy (comparison with method using high-performance liquid chromatography (HPLC)) were excellent. This method was more specific than with HPLC, so that the recovery rate of sugars was improved. In the absence of a significant matrix effect, purification of the extract was unnecessary, thus simplifying the procedure and contributing to its robustness. A micro-method is thus proposed which can be applied to 5 mg of plant powder. This miniaturization affects neither the precision of the MP method nor sugar concentrations. The results of starch assays further demonstrated that this micro-method was appropriate to the analysis of NSC in small samples of woody plant tissues. Reliable, rapid and simple to perform, this micro-method is less expensive than HPLC or other classic enzymatic methods. (c) 2007 Society of Chemical Industry.