<p>Innate immune response modulating impurities (IIRMI) with adjuvant potential have emerged as important factors in the immunogenicity risk assessment of protein, peptide, and oligonucleotide therapeutics, particularly for follow-on products where minimal or no clinical studies are available. To assess the impact of differences in impurities on specific cell types, we developed a new IIRMI assay termed multiplexed immunophenotyping for innate activation assessment (MIIAA) that employs spectral flow cytometry to capture single-cell responses to drug products and potential impurities. This technique introduces a new live fluorescent cell barcoding platform that enables sample multiplexing for homogeneous staining with a single fluorescent antibody cocktail composed of identity and activation markers that are acquired simultaneously with a five laser Cytek Aurora. Samples are digitally reassigned to their original testing conditions by positive and negative gating of barcode dyes. Cellular subsets are identified by dimensionality reduction of surface markers with UMAP then gated using cell-specific markers. Here we use trace levels of TLR3, 7/8 and 9 agonists (Poly(I:C), R848, and CpG ODN) to characterize specific responses in B cells, monocytes, cDC and pDC. Importantly, MIIAA captures single-cell responses to nucleic acid impurities in the presence of therapeutic oligonucleotides or monoclonal antibodies with high sensitivity. Taken together, MIIAA offers a powerful immunophenotyping tool to characterize single-cell responses to drug products and potential immunomodulatory impurities that may find utility in drug pipelines to characterize the impact of therapeutics on specific immune cells and to interrogate immunogenic or immunomodulatory risk in comparisons between reference and follow-on products.</p> Graphical Abstract <p></p>