Previously we presented a platform for performing high throughput pretreatment and enzymatic hydrolysis of lignocellulosic material utilizing a custom designed and manufactured 96-well plate steam reactor system. While useful in comparative studies within defined sample sets, conversion yields and extents cannot be assessed without a concurrent high-throughput compositional analysis of the starting material. The determination of the structural carbohydrate content of lignocellulosic materials is a crucial experimental step in the study of lignocellulose recalcitrance and the conversion to fuels and chemicals. Typical protocols require cumbersome equipment and time consuming steps to produce even adequately accurate results. Building on our earlier high-throughput pretreatment platform, we have expanded our capabilities to include a 96-well method for determining the key structural carbohydrates present in lignocellulosic materials. The system utilizes a deeper (~1.7 mL) 96-well Hastalloy reactor to perform a scaled down version of the most commonly cited two-stage sulfuric acid hydrolysis protocol. Rapid determination of glucose and xylose released by the process is performed via glucose oxidase and xylose dehydrogenase coupled assays, while NMR and mass spectroscopy are being evaluated for rapid assessment of other sugars and components. We feel the introduction of such technology will rapidly accelerate the pace of substrate-based studies in the lignocellulose recalcitrance field.