Objective: The identification of proteomic signatures in treatment-naïve individuals newly diagnosed with inflammatory bowel disease (IBD) holds promise for elucidating the pathophysiological mechanisms underlying IBD and to differentiate Crohn´s disease (CD) and ulcerative colitis (UC), which could aid in early diagnosis and targeted therapeutic interventions. Design: A discovery phase was performed using label-free quantitative proteomics to identify proteomic profiles in serum extracellular vesicles (EVs), serum, urine and intestinal tissue from 100 newly diagnosed IBD patients (50 CD and 50 UC), and 51 healthy controls (HC). Candidate biomarkers were evaluated by ELISA on serum from an independent cohort of 87 CD, 134 UC and 99 HC (validation cohort) and confirmed by immunohistochemistry on biopsies from the same discovery phase cohort. Results: We identified 419 proteins in serum EVs, 468 in serum, 683 in urine, and 2,603 in intestinal tissue. ELISA results showed lower levels of TTR and APOC3 and higher levels of ATRN in UC patients compared to HC. Comparison of potential biomarkers between CD and HC groups showed lower levels of TTR and higher levels of ATRN. S10A9 was differentially upregulated in CD compared to UC. The expression of PRDX4 and AZU1 was increased in ileum from CD patients compared to HC, whereas AOFB was lower in the CD ileum and CD and UC left colon compared to HC tissues. Conclusion: Comprehensive proteomic analysis has pinpointed key IBD associated proteins that may help elucidate the mechanisms underlying IBD pathogenesis. Altered molecular pathways underlying IBD should be explored in future research.