Conventional wet chemistry methods to determine biomass composition are labor- and time-intensive and require larger amounts of biomass (300 mg) than is often available. To overcome these limitations and to support a high-throughput pretreatment and hydrolysis (HTPH) screening system, this article reports on the development of a downscaled biomass compositional analysis that is based on conventional wet chemistry techniques but is scaled down by a factor of 100 to use significantly less material. The procedure is performed in readily available high-performance liquid chromatography vials and can be automated to reduce operator input and increase throughput. Comparison of the compositional analyses of three biomasses determined by the downscaled approach to those obtained by conventional methods showed that the downscaled method measured statistically identical carbohydrate compositions as standard procedures and also can provide reasonable estimates of lignin and ash contents. These results demonstrate the validity of the downscaled procedure for measuring biomass composition to enable the calculation of sugar yields and determination of trends in sugar release behavior in HTPH screening studies. Biotechnol. Bioeng. 2011;108: 306-312. (C) 2010 Wiley Periodicals, Inc.