The purpose of this study was to develop an in-vitro digestion protocol to evaluate the antioxidant potential of the peptides found in processed cheddar cheese using digestion enzymes. We studied first antioxidant and angiotensin converting enzyme (ACE) inhibition and antioxidant activities of processed cheddar cheese with the addition of spices e.g. cumin, clove and black pepper made from buffalo milk and ripened for 9 months. Then we conducted an in vitro digestion of processed cheddar cheese by gastric and duodenal enzymes. Freeze dried water (WSE) and ethanol soluble fractions (ESE) of processed cheddar cheese were also monitored for their ACE inhibition activity and antioxidant activities. In our preliminary experiments, different levels of spices (cumin, clove and black pepper) were tested into cheese matrix and only one level 0.2g/100g (0.2%) on the basis of cheese weight was considered good concerning sensory evaluation. Significant increase in ACE-inhibition (%) of processed Cheddar cheese as well as its WSE and ESE was obtained. Lower IC50 values were found after duodenal phase digestion compared to oral phase digestion.