Pitaya fruit processing generates a large amount of pitaya peel which contains high level of dietary fiber, betacyanins and phenolic compounds. In this study, pitaya peel powder was added to cookie formulation and the ratio of pitaya peel powder was varied from 0 to 25% of the amount of wheat flour. The obtained cookie samples were then used for analysis of proximate composition, physical properties, overall acceptability, predicted glycemic index and antioxidant release during in vitro sequential digestion. The higher the ratio of pitaya peel powder in the recipe, the greater the dietary fiber, betacyanin and phenolic contents and antioxidant activities of the product. High pitaya peel ratio also resulted in enhanced hardness and increased intensity of red color of the cookies; however, all cookie samples were considered acceptable. The use of pitaya peel significantly reduced the glycemic index of cookies and all supplemented pitaya peel cookies were classified as having low glycemic index. The release of betacyanins, phenolics and antioxidant activities from the cookie samples was recorded at the salivary, gastric, intestinal and colon steps during in vitro sequential digestion and the gastric digestion showed the highest release of antioxidant content and activity. The increased pitaya peel ratio in the cookie recipe improved the antioxidant activities of the soluble fractions at the four digestive steps.