Abstract Robust pharmacodynamic (PD) assays are valuable to confirm putative drug mechanisms and downstream effects in support of early phase clinical trials. Evolving methods to achieve consistent performance across patients, clinical trials, and laboratories is critical for the success of clinical pharmacodynamic measurements. We have developed integral quality control tools to support clinical implementation of quantitative multiplex immunofluorescence assays (qmIFAs) that include xenograft tissue reference calibrators. These reference slides help to provide confirmation that the pharmacodynamic assay is reporting biomarker levels within designated target ranges with expected subcellular localization, helping to control for run-to-run issues with the assay procedures, reagents, or equipment; however, significant challenges are faced in producing these key reagents. Compared to conventional cell pellets, xenograft controls require more time, expense and specialized facilities; however, the tumor tissues are much more representative of the heterogeneity found in clinical biopsies. The challenges associated with xenograft controls include tissue and biomarker variability attributed to necrotic areas, fixation artifacts, and variable drug distribution. During our qualification process, xenografts undergo multiple rounds of selection and analysis to identify specimens that exhibit good tissue quality, adequate regions of interest (ROI), and appropriate target signal levels to support the intended biomarker measurements. Slide lots are produced, qualified, and stored in a manner that ensures adequate lot sizes, robust and reproducible slide to slide performance, and appropriate shelf life. Xenograft tissue reference standards representing a range of basal or drug-induced biomarker expression in appropriate models have been developed for several informative PD markers including Epithelial Mesenchymal Transition (EMT) markers such as E-Cadherin, Vimentin, and Beta-Catenin; DNA damage response markers γH2AX, pNBS1 and Rad51; apoptosis induction markers for colocalized γH2AX/cleaved Caspase 3; and the cell cycle markers pY15cdk and pHH3 among others and have been used as important tools to support ongoing clinical pharmacodynamic analyses of research biopsies at the NCI and other institutions (PMIDs 25964244, 29682208, 30792217, 31732654). In summary, generalized methods and workflows that will be made public have been developed by the NCI to optimize the production of qualified reference slide lots of sufficient reproducibility, stability, and quantity to support ongoing NCI-supported clinical trial pharmacodynamic assays for several key biomarkers. Funded by NCI Contract No HHSN261200800001E. Citation Format: Gabriel J. Benton, Katherine V. Ferry-Galow, Victor E. Lonsberry, Manisha Mohandoss, Howard Stotler, Debbie Trail, Kyle Georgius, Melinda Hollingshead, Deborah Wilsker, Robert Kinders, James H. Doroshow, Ralph E. Parchment. Development of robust and reproducible controls to support clinical implementation of quantitative immunofluorescence assays for pharmacodynamic biomarkers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6495.