Rheumatoid factor are autoantibodies that are found in +/- two thirds of patients with rheumatoid arthritis, a chronic autoimmune disease characterized by inflamed, swollen joints. They consist of polyclonal antibodies that target the Fc part of IgG. The antibodies are not highly specific for rheumatoid arthritis and clinical assays to measure rheumatoid factor are poorly harmonized. We studied rheumatoid factor using a mass-spectrometry-based approach in seropositive, seronegative rheumatoid arthritis patients and in disease controls. Rheumatoid factor was captured on Fc coated microwell plates, isolated, digested into peptides and analyzed by liquid chromatography tandem mass spectrometry. Principal component analysis and sparse partial least squares discriminant analysis showed that peptides derived from seropositive rheumatoid arthritis patients clustered away from the controls. Mass spectrometry analysis revealed framework region-derived and variable region-derived peptides that were enriched in rheumatoid factor positive sera. However, mass spectrometry de novo sequencing failed to sequence the majority of unidentified peptides. Furthermore, mass spectrometry analysis revealed different rheumatoid factor isotypes. In addition to IgM, also IgA and IgG isotypes were observed. In conclusion, mass spectrometry is able to capture the complexity and isotypes of rheumatoid factor autoantibodies, but advances in de novo sequencing are needed to fully characterize the variable part of the antibodies.