Updated project metadata. Mass spectrometry (MS)-based immunopeptidomics is an attractive antigen discovery method with growing clinical implications, especially for personalized cancer immunotherapies. However, the current experimental approach to extract HLA-I-restricted peptides requires a bulky sample source, which remains a challenge for obtaining clinical tumor samples. We present an innovative streamlined workflow that requires a low sample volume. The workflow integrates the immunoaffinity purification (IP) and C18 peptide cleanup steps on a single microfluidics platform with automated liquid handling and minimal sample transfers, resulting in higher assay sensitivity. We also demonstrate how state-of-the-art data-independent acquisition (DIA) MS method further enhances the depth and reproducibility of tandem mass spectrometry (MS2) spectra-based peptide sequencing. As a result, over 4,000 and 5,000 HLA-I restricted peptides can be reliably identified from as low as 0.2 million human RA957 cells and a melanoma tissue of merely 5 mg, respectively. We also identified multiple known immunogenic tumor-associated antigens and hundreds of peptides derived from non-canonical protein sources. The presented workflow is a powerful tool for identifying the immunopeptidome of sparse samples.