The efficacy of 5-aminosalicylic acid (5-ASA) as the first-line therapy for ulcerative colitis (UC) is determined by the extent of drug delivery to the inflamed region. Moreover, differences among the various formulations influence delivery of the drug. In this study, we examined the clinical significance of colonic mucosal concentrations of 5-ASA and N -acetylmesalamine (Ac-5-ASA) in UC patients receiving a pH-dependent or time-dependent release formulation of 5-ASA. The subjects were 67 patients with UC who were treated with a pH-dependent or time-dependent formulation of 5-ASA between December 2011 and April 2014. A retrospective observational analysis of clinical outcomes was performed using the clinical activity index (CAI) obtained on the day of biopsy. Colonic mucosal concentrations of 5-ASA and Ac-5-ASA in biopsy samples were measured by LC-tandem mass spectrometry/mass spectrometry. Patients who were treated with the pH-dependent formulation had higher colon mucosal concentrations of 5-ASA than those who were treated with the time-dependent formulation. Additionally, 5-ASA concentration was significantly higher in patients with CAI scores ≤3. A higher concentration of Ac-5-ASA was achieved with the time-dependent formulation than with the pH-dependent formulation. Furthermore, patients with CAI scores ≤3 had higher concentrations of 5-ASA than those with CAI scores ≥4. The colonic mucosal concentration of 5-ASA in patients with UC is influenced by the pharmaceutical formulation and the remission status of UC.