BACKGROUND: Upadacitinib is a selective Janus kinase (JAK) 1 inhibitor approved by the Food and Drug Administration for the treatment of moderate-to-severe inflammatory bowel disease (IBD). We aimed to establish and validate a method for determining Upadacitinib in patients with IBD by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. METHODS: The mobile phase was 0.1 % formic acid: acetonitrile (35:65, v/v) at a flow rate of 0.40 mL/min. Upadacitinib and its internal standard Upadacitinib (15)N, d(2) were separated by a Waters Xbridge BEH C18 column (4.6 x 100 mm, 2.5 mum) and subjected to mass analysis using positive electrospray ionization (ESI). RESULTS: The calibration range of Upadacitinib was 0.5-200 ng/mL with the correlation coefficient r(2) >/= 0.99. Accuracies ranged from -9.48 % approximately 8.27 % and the inter- and intra-day precisions were less than 15 % for all analytes in quality control samples. There was no significant matrix effect. The range of extraction recoveries was 87.53-93.47 % for all analytes. Twenty-one plasma samples were obtained from the sixth affiliated hospital of Sun Yat-sen University. The median plasma concentration of Upadacitinib was 7.32 (0.56-26.78) ng/mL. CONCLUSION: This newly developed method is sensitive, simple, and successfully applied in determining Upadacitinib in IBD patients to provide reference for safe and effective drug administration in clinical practice.