Introduction: Rheumatoid arthritis (RA) is an autoimmune disease resulting from a response driven by self-reactive CD4+ T cells that recognize autoantigenic peptides presented by antigen-presenting cells (APCs). Recent evidence suggests that CD8+ T cells are also important players in this process. This study aims to define the immunopeptidome of HLA class I molecules from RA synovial tissue (ST)- APCs and synovial fluid (SF)-pulsed monocyte-derived dendritic cells (DCs), and to prove the suitability of this approach to identify peptides recognized by CD8+ T cells from RA patients. Methods: HLA-ABC/peptide complexes were obtained from DCs generated from healthy subjects (HS), which were pulsed with a pool of RA SF (SF-DCs) or left unpulsed (UP-DCs), or obtained directly from RA ST. Isolated peptides were sequenced by mass spectrometry. The autoantigenicity of a set of ten peptides selected from this repertoire was estimated by their ability to activate CD8+ T cells from RA patients, as measured by the induction of intracellular IFN-γ expression and surface exposure of CD107a by flow cytometry. Results: Between 107 to 663 peptides were obtained from DC samples, while over 3,500 class I peptides were identified from each ST sample. The number of peptides was narrowed down based on prioritization steps that included the selection of sequences derived from RA-relevant, immune-related proteins, for further CD8+ T-cell stimulation assays. The frequencies of CD8+ T cells co-stained for IFN-γ and CD107a were significantly higher in RA patients than in HS in response to peptides derived from the proteins MIF, ETS1, USF1, VIM, and AHR. Discussion: In the present work, we validated the use of an immunopeptidomic strategy to identify a series of novel autoantigenic CD8+ T-cell epitopes for RA, derived from synovial, mostly immune-related proteins, which may be useful for future clinical applications.