Glycosyl hydrolases (GH) were isolated and purified from various fungal sources to rationally design enzyme cocktails with higher specific activity (compared to commercially available cellulase blends) on ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were cellobiohydrolase I (CBH I, GH family 7A), cellobiohydrolase II (CBH II, GH family 6A), endoglucanase I (EG I, GH family 7B) and β-Glucosidase (βG, GH family 3). The two core hemicellulases were an endoxylanase (EX, GH family 11) and β-xylosidase (βX, GH family 3). Enzyme family and purity were confirmed by proteomics. Synergistic interactions among the six core enzymes for varying relative and total protein loadings (8.25, 16.5 and 33 mg/g glucan) during hydrolysis of AFEX-treated corn stover were investigated using a high-throughput microplate based assay. The optimal composition (based on % protein mass loading) of the core six enzyme mixture was CBH I (28.4%), CBH II (18.0%), EG I (31.0%), EX (14.1%), βG (4.7%) and βX (3.8%). Other accessory enzymes such as endo-xylanases (GH 10), α-arabinofuranosidases (GH 51, 54, 62) and α-glucuronidase (GH 67) were doped into the core six enzymes cocktail to identify critical synergistic interactions. Several hemicellulases were found to play a synergistic role in enhancing both xylan and glucan hydrolysis yields for AFEX treated corn stover. These results demonstrate a rational strategy for the development of a minimal, synergistic glycosyl hydrolase cocktail that could reduce enzyme usage and maximize fermentable sugar yield from pretreated lignocellulosics.