Abstract The human immune system is regulated by a complex network of inhibitory and stimulatory immune checkpoint receptors that regulate T Cell function. These receptors are promising new immunotherapy targets for the treatment of a variety of cancers and autoimmune disorders. Novel therapies employing a variety of strategies have been developed that rely on releasing the brakes on the immune system, with the aim of eliminating tumor cells even more efficiently. Immunotherapies designed to block co-inhibitory receptors (e.g. PD-1, CTLA-4) are showing unprecedented efficacy in the treatment of cancer. However, not all patients and tumor types respond to this approach. This has resulted in broadening of immunotherapy research programs to target additional co-inhibitory (e.g. LAG-3, TIM-3, TIGIT) and co-stimulatory (e.g. 4-1BB, GITR, OX40, ICOS) receptors, individually and in combination. A major challenge in the development of antibody-based biologics is access to quantitative and reproducible functional bioassays. Existing methods rely on primary cells and measurement of complex functional endpoints. These assays are cumbersome, highly variable and fail to yield data required for drug development in a quality-controlled environment. To address this need, we have developed a suite of cell-based functional bioassays to interrogate modulation of immune checkpoint receptors individually (e.g. PD-1, LAG-3, TIM-3, GITR, 4-1BB) and in combination (e.g. PD-1+CTLA-4, PD-1+LAG-3). These bioassays consist of stable cell lines that express luciferase reporters driven by response elements under the precise control of mechanistically relevant intracellular signals from TCR and immune checkpoint receptors. Thus, these bioassays reflect mechanisms of action for drug candidates designed for each immune checkpoint receptor and demonstrate high specificity, sensitivity and reproducibility. The bioassays are prequalified according to ICH guidelines and show the precision, accuracy and linearity required for routine use in potency and stability studies. Here we describe the application of MoA-based immune checkpoint receptor bioassays as tools for biologics drug discovery, development, potency and stability studies. Citation Format: Jamison Grailer, Julia Gilden, Pete Stecha, Denise Garvin, Jun Wang, Michael Beck, Jim Hartnett, Gopal Krishnan, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Quantitative cell-based reporter bioassays for functional characterization of immunotherapeutic biologics targeting co-stimulatory and co-inhibitory immune checkpoint receptors [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 944.