Abstract Dendritic cell (DC) based immunotherapy is a promising treatment alternative for various solid tumor and hematological malignancies. DCs are capable of initiating and propagating antigen-specific adaptive immune responses through their intrinsic maturation process. Identifying novel, effective adjuvant drug candidates that can stimulate DC maturation while understanding and accounting for the variability of patient-specific DC cell responses to the immunostimulatory adjuvants is essential for development of such immunotherapies. Reliable, high throughput assay platforms that characterize patient-specific modulatory effects during the DC maturation process in vivo and in vitro are also highly desirable. In this study, we present a high throughput in vitro primary cell assay to evaluate dose-dependent DC maturation in a panel of human leukocyte antigen (HLA) typed individual donors. CD14+ monocytes from four healthy donors were isolated from leukapheresis derived peripheral blood mononuclear cells (PBMC) and cultured for seven days with GM-CSF and IL-4 to achieve differentiation to DC. Cells were then stimulated for 24 hours with lipopolysaccharide (LPS), CpG oligonucleotides, or nine different novel toll-like receptor 4 (TLR4) ligands at three concentrations. To quantify the differentiation of monocytes into DC and subsequent maturation, flow cytometry was used to measure the expression of biomarkers, MHC-II, CD40, CD80, CD83, and CD86. Greater than 99% MHC-II expression was observed across all donors and confirmed successful differentiation to DC. The TLR4 ligands showed reduced biomarker expression, as compared to pathogenic E. coli LPS but similar to PHAD, a synthetic TLR4 agonist currently used in adjuvants. While monocytes from all donors responded to the ligands tested, the intensity of activation marker expression varied among donors with LPS/TLR4 ligand induced maturation process showing 43-91% CD40 and 27-82% CD83 expression, highlighting the necessity of multi-donor screening for drug development. In conclusion, a high throughput in vitro primary monocyte derived DC differentiation assay has been developed and is commercially available to assist researchers in characterizing donor-specific DC responses to immunostimulatory adjuvants. Citation Format: Qin Chen, Anthony Lawrenz, Alan Wang, Erin Kelly, Erin Harberts, Robert K. Ernst, Jay Tong. Donor-specific primary monocyte-derived dendritic cell maturation model for high-throughput screening of immunostimulatory adjuvants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4021. doi:10.1158/1538-7445.AM2017-4021