The challenge of converting cellulosic biomass to sugars dominates the cost of biological processing to ethanol and other products. The BioEnergy Science Center (BESC), led by Oak Ridge National Laboratory, is addressing this challenge by advancing plants to reduce recalcitrance and new biocatalysts to improve deconstruction. Systems biology in conjunction with new high-throughput (HTP) analytical and computational technologies will be instrumental to this approach. In order to enable the screening of the many different natural and genetically modified biomass types and biocatalysts and identify those with enhanced features for biological conversion, a new HTP tool for pretreatment and enzymatic hydrolysis system is being developed. In our approach, pretreatment with dilute acid or just water is integrated with enzymatic hydrolysis in the same 96 well plate. Each well is loaded with 2 – 5 mg of milled biomass that is suspended in water or dilute acid to a final slurry concentration of 1 - 2% w/w. After loading, the plate is sealed and rapidly heated to the target pretreatment temperature in a steam chamber and held there for the desired reaction time. The pretreatment reaction is then quenched by flooding the steam chamber with cold water. Samples which were pretreated with dilute acid are neutralized in situ, and citric acid buffer, sodium azide, and enzymes (cellulase and xylanase) are directly added to each well, with the enzyme loading based on the original glucan and xylan content of the raw biomass. Next, the reactor is re-sealed and placed in an incubator shaker at 50°C for 72 hours, and the hydrolyzate sugar concentration in each well is analyzed using a redox enzyme-mediated spectroscopic assay. Applications of this integrated approach will be presented for poplar wood and switchgrass.