The downstream processing of biotechnological products from fermentation broth is an important step of production and development of cost effective, efficient downstream processing of many biotechnological products. The present study was conducted by employing aqueous two phase systems (ATPSs) for the extraction of cephalosporin C (CPC) from whole fermentation broth of Acremonium chrysogenum. The biphasic system was prepared by mixing equal aliquots of 15% w/w polyethylene glycol (PEG) 3350 with 15% (NH4)2SO4. The effects of pH, neutral salts, temperature and centrifugal force on partitioning in ATPS to develop efficient extraction system for recovery of CPC from fermentation broth were also examined. The extraction efficiency was improved by enhancing the centrifugal force. Similarly centrifugation for 12.5 min also gave the maximum extraction. Improvement in the recovery yield was also observed by the addition of 0.1% NaCl. The concentration of CPC was determined by high performance liquid chromatography (HPLC). Slight modifications in the mobile phase from 10 to 5% MeOH improved CPC resolution. Further development of more inexpensive systems for extraction can be the future target of research.Keywords: Cephalosporin C, Acremonium chrysogenum, fermentation, aqueous two phase system (ATPS