The dinitrosalicylic acid (DNS) method is routinely used to estimate the concentration of reducing sugars in biomass hydrolysates. To decrease liquid handling times and allow automation, scaled versions of the original DNS method have been created. However, reaction volumes, incubation times and reading wavelengths have not been optimised for this new format. Here we show how these parameters affect assay performance. On the basis of these results we recommend the use of 1:20, sample: DNS reagent, to analyse hydrolysates containing 0-100 g L (1) reducing sugars and heating in a thermocycler (100 degrees C, 1 min) before quantification - reading at 540 or 580 nm and selecting the most appropriate wavelength based on sample absorbance. This method is significantly faster and more precise than previous methods and eliminates the need for sample dilution. Hydrolysates from enzymatically saccharified, steam-exploded wheat straw were analysed to demonstrate the improved precision of this method when using complex substrates. (C) 2012 Elsevier Ltd. All rights reserved.