The automated 96-well microplate method is a rapid hydrolytic assay technique (essentially a scaled down version of the NREL LAP 009 protocol) developed at our laboratory. The critical bottlenecks addressed in the development of this technique are: reproducible substrate delivery, minimization of evaporation losses, hydrolysis time period, rapid sugar assay and high throughput. A suitable method for delivery of substrate was through dispensing a biomass slurry suspension, using a robotic liquid handling workstation. Delivery parameters were optimized for varying substrate slurry concentrations. The microplate method was optimized for hydrolysis of avicel and AFEX treated corn stover using a mixture of commercial cellulase and β-glucosidase. The method was validated and statistical analysis on the data was performed. We also screened several commercially available cellulases, xylanases and beta-glucosidases under a wide range of total enzyme concentrations for the hydrolysis of AFEX pretreated biomass. Further screening of new fungal cellulase systems that aid synergistic hydrolysis of AFEX treated biomass was also accomplished using the microplate assay technique.