Chondroitinases are useful in treating cancer and spinal cord injuries. The work reported so far does not focus on production of fungal chondroitinase. The present study focuses on the optimization of cultural conditions using design of experiments for production of chondroitinase by a fungal isolate CHCS-2, obtained from humus containing poultry waste. The cultural conditions were optimized systematically using a one-factor-at-a-time (OFAT) approach, factorial design, Plackett-Burman design (PBD) and Box- Behnken design to identify the composition of the production medium and the level of the variables. The level of significance of the medium components selected by OFAT study viz., yeast extract, chondroitin, and phosphate, was analyzed by factorial studies. The effect of eleven minerals on the enzyme yield was studied by PBD. The interaction among the medium components selected using factorial and PBD viz., yeast extract, cobalt and aluminum was investigated using the Box-Behnken design (BBD). Under the optimized conditions, maximum chondroitinase yield was 22.5 U/mL.