Abstract The classical MHC Class II molecules HLA-DP, DQ and DR proteins bind antigenic peptides in the endosomes of APCs and present them to CD4+ T cells whereas HLA-DM and DO are the non-classical proteins. DM catalyzes the exchange of CLIP peptides with other endosomal peptides whereas DO acts as an inhibitor of DM activity. The clear biochemical effect of DO in inhibiting DM is reported but its effect on the peptide repertoire is still not clear. We used Quadrupole Orbitrap mass spectrometry for characterizing peptides presented by the MHC Class II protein DR1 from CRISPR/Cas9 deleted LG2 cells. We identified 6139 peptides in wild type cells (WT) but only 5227 peptides from DO−/− cells (DO-KO). The label free quantitation analysis of WT Vs DO-KO showed DM sensitive CLIP peptides were present in lower abundance in DO-KO, consistent with cell surface staining experiments. We analyzed peptide diversity in the eluted peptidome using diversity measures like Shannon’s Entropy, Simpson’s diversity and Berger-Parker Index. In each case the diversity was higher for the WT peptidome as compared to DO-KO. We screened several peptides for binding affinity and DM susceptibility by analyzing their relative abundances in WT and DO-KO. Peptides from group WT > DO-KO, WT only showed lower binding affinity and high DM sensitivity as compared to peptides in DO > WT, DO only. The same phenomenon was observed for peptides eluted from I-Ab purified from B cells and thymus of BL/6 as compared to H2-O−/− mice. These results indicate that DO/H2-O broadens the peptide repertoire whereas its absence restricts the repertoire resulting in presentation of fewer different epitopes, and highlights the role of DO in regulating the MHC-II peptide repertoire by modulating DM peptide editing.