In this work, six core glycosyl hydrolases (GH) were isolated and purified from various sources to help rationally optimize an enzyme cocktail to digest ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were endoglucanase I (EG I, GH family 7B), cellobiohydrolase I (CBH I, GH family 7A), cellobiohydrolase II (CBH II, GH family 6A) and β-glucosidase (βG, GH family 3). The two core hemicellulases were an endo-xylanase (EX, GH family 11) and a 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 loading (8.25, 16.5 and 33 mg/g glucan) during hydrolysis of AFEX-treated corn stover was studied using a high-throughput microplate based protocol. The optimal composition (based on% protein mass loading) of the cocktail mixture was CBH I (28.4%): CBH II (18.0%): EG I (31.0%): EX (14.1%): βG (4.7%): βX (3.8%). These results demonstrate a rational strategy for the development of a minimal, synergistic enzymes cocktail that could reduce enzyme usage and maximize the fermentable sugar yields from pretreated lignocellulosics. © 2009 Elsevier Ltd.