Experimental designs have been applied for optimizing the process parameters for hydrogen production from maize stalk hydrolyzate by a newly isolated facultative strain. Using Plackett-Burman design glucose, yeast extract, malt extract, peptone, and NaCl have been identified as significant variables influencing hydrogen production and these variables are subsequently optimized using a central composite design (CCD). Box-Behnken design has been adopted for optimizing the process parameters for maximum hydrogen production. Using this statistical optimization method a maximum hydrogen yield of 0.91 mol H-2/mol glucose has been achieved.