Mesalamine is a standard first-line therapy for managing chronic inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis. Despite its established efficacy, the precise mechanism of action within enterocytes remains unclear. This study aimed to develop and validate Physiologically Based Biopharmaceutics Models (PBBM) for mesalamine (5-ASA) and its metabolite, acetyl mesalamine (Ac-5-ASA), to predict drug concentrations in plasma, colonic lumen, and colonic tissue of healthy subjects and compare the results to measured concentrations. Using the Simcyp Simulator (V22), the models accurately predicted plasma concentrations for various formulations, including intravenous, oral immediate-release and controlled release formulations within a two-fold range. Results also captured the intestinal and hepatic metabolism converting mesalamine to acetyl mesalamine. However, significant discrepancies were observed in predicting luminal and tissue concentrations, with underpredictions for Claversal and Pentasa formulations reaching factors of up to 506 and 55 for 5-ASA and Ac-5-ASA in colonic tissue, respectively. These discrepancies highlight limitations in current modeling approaches, particularly in simulating drug accumulation within enterocytes. Despite these challenges, this investigation highlights both the potential benefits and the complexities of using PBBMs. Future work should focus on generating definitive N-acetyl transferase (NAT1) abundance data with an in-vitro in-vivo extrapolation link, improving approaches to better explore local drug concentrations in the gastrointestinal tract, and addressing the gap in accurately predicting luminal and tissue concentrations in the colon.