This work focuses on tracking ulcerative colitis in mice. High labeling yield and radiochemical purity were achieved for the formation of a [125/131I]balsalazide radiotracer at optimum conditions of oxidizing agent content (chloramines‐T [Ch‐T], 75 μg), substrate amount (100 μg), pH of reaction mixture (6), reaction time (30 min), and temperature (37°C), using radioactive iodine‐125 (200–450 MBq). The radiolabeled compound, [125/131I]balsalazide, was stable in serum and saline solution during 24 h. Balsalazide is acting as a peroxisome proliferator‐activated receptor (PPARγ). Biodistribution studies were carried in normal and ulcerated colon mice. High uptake of 75 ± 1.90% injected dose/g organ (ID/g) observed in ulcerated mice confirmed the suitability of [131I]balsalazide as a novel radiotracer for ulcerative colitis imaging in mice.