<jats:title>Abstract</jats:title><jats:sec><jats:title>Background and Aim</jats:title><jats:p>5‐Aminosalicylic acid (5‐ASA) is a fundamental treatment for mild‐to‐moderate ulcerative colitis (UC). 5‐ASA is taken up into the colonic mucosa and metabolized to <jats:italic>N</jats:italic>‐acetyl‐5‐ASA (Ac‐5‐ASA). Few studies have assessed whether mucosal 5‐ASA and Ac‐5‐ASA concentrations are associated with endoscopic remission. This study aimed to investigate differences in 5‐ASA and Ac‐5‐ASA concentrations according to endoscopic activity.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>This single‐center, prospective, cross‐sectional study was conducted between March 2018 and February 2019. UC patients who were administered with 5‐ASA medication for at least 8 weeks before sigmoidoscopy were enrolled. Mucosal 5‐ASA and Ac‐5‐ASA concentrations were measured using liquid chromatography with tandem mass spectrometry. The primary endpoint was defined as the difference in mucosal concentrations of 5‐ASA and Ac‐5‐ASA, according to the Mayo endoscopic subscore (MES).</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Mucosal concentrations were analyzed in 50 patients. In the sigmoid colon, the median 5‐ASA concentration in patients with MES of 0 (17.3 ng/mg) was significantly higher than MES ≥ 1 (6.4 ng/mg) (<jats:italic>P</jats:italic> = 0.019). The median 5‐ASA concentrations in patients with Ulcerative Colitis Endoscopic Index of Severity ≤ 1 (16.4 ng/mg) were also significantly higher than in patients with Ulcerative Colitis Endoscopic Index of Severity ≥ 2 (4.63 ng/mg) (<jats:italic>P</jats:italic> = 0.047). In the sigmoid colon, the concentration of Ac‐5‐ASA was higher in patients with MES of 0 (21.2 ng/mg) than in patients with MES ≥ 1 (5.81 ng/mg) (<jats:italic>P</jats:italic> = 0.022).</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>The present study showed that mucosal Ac‐5‐ASA concentrations, as well as 5‐ASA concentrations, are higher in UC patients with endoscopic remission. Ac‐5‐ASA may be useful for a biomarker of 5‐ASA efficacy.</jats:p></jats:sec>