Type II collagen (CII) is the most abundant protein in joint cartilage. Antibodies to CII appear around the clinical onset of the autoimmune disease rheumatoid arthritis (RA) in a subset of patients. They target specific epitopes on CII and can be pathogenic or protective. Assays for early detection of such autoantibodies may provide new opportunities for selecting effective treatment strategies of RA. We report the efficient and reproducible assembly of an array of covalently branched native and citrullinated triple helical peptides (THPs) from CII that contain defined autoantibody epitopes. Both monoclonal antibodies and sera from experimental mouse models show a unique reactivity toward the THPs, compared to cyclic peptides containing the epitopes, revealing the importance that the epitopes are displayed in a triple-helical conformation. Importantly, antibodies against three of the THPs that contain major CII epitopes were found to be increased in sera from patients with RA, compared to control persons. These results indicate that such synthetic THPs should be included in multiplex analysis of autoantibodies that are uniquely occurring in individuals with early RA, to provide valuable information on disease prognosis and on what type of therapy should be chosen for individual patients.