Abstract The objective of this research was to develop an in vitro dendritic cell based assay to assess if differences in internalization of biotherapeutic drugs (BioTx) and/or intra-cellular trafficking could be measured and used to help predict clinical immunogenicity risk. An unwanted immune response can adversely impact pharmacokinetics, safety and efficacy. Since immunogenicity in preclinical species is not clinically predictive there is a strong need for in vitro tools to enable BioTx drug design. Immature dendritic cells (iDCs) were selected because of their high fluid phase pinocytosis rates, ability to recycle IgGs and route immunogenic molecules to MHC-II compartments. Initial assay development evaluated iDC differentiated from a monocytic human cell line vs primary monocytes from fresh whole blood using IL4 and GM-CSF. Fluid phase internalization rates of Ovalbumin and Lucifer yellow decreased from day 3 to day 5 and CD80, CD86 and HLA-DR expression increased consistent with DC maturation. Imaging cytometry was used to measure internalization extent, rate and LAMP-1 co-localization of fluorescently conjugated ovalbumin and BioTx with known clinical immunogenicity incidence. Primary monocytes derived iDC were selected based on extent of internalization for further assay characterization in multiple donors. No target expression was observed. The extent of Adalimumab, Traztuzumab, and Utomilumab internalization was correlated with clinical immunogenicity incidence suggesting the assay may be able to detect frame work differences. Statistical power calculation of fold change set required donor numbers at 10. The assay can be used in combination with other in vitro immunogenicity assays to support BioTx design.