TIM-3 (CD366, HAVCR2) is an immune checkpoint receptor expressed on a subset of activated T cells, regulatory T cells (Tregs), macrophages and dendritic cells. Preclinical studies have shown that TIM-3 signaling regulates Th1 cytokine responses, and that expression of TIM-3 is associated with resistance to autoimmunity. Consistent with these observations, blockade of TIM-3 leads to tumor rejection in mouse models of colorectal and ovarian cancer. While the in vivo effects of TIM-3 are clear, the cellular events leading to these outcomes are not understood. There is a growing body of literature suggesting a context- and cell type-dependent role for TIM-3 in T-cell activation that is more complex than reported previously. It is now evident that TIM-3 has the capacity to inhibit or costimulate T-cell receptor (TCR) signaling in different in vitro systems. Moreover, the identities and functional relevance of ligands for TIM-3 have long been the subjects of intense scientific debate. Phosphatidylserine (PS), galectin-9, CEACAM-1 and others have been proposed as TIM-3 ligands, adding another layer of complexity to TIM-3 signaling. The TIM-3 Bioassay (Cat.# JA2211, JA2215) is a bioluminescent reporter cell-based assay that overcomes the limitations of existing assays and can be used to measure the potency and stability of antibodies and other biologics targeting TIM-3. The assay consists of two cell lines: TIM-3 Effector Cells (Human T cells genetically engineered to express human TIM-3 and a NanoLuc luciferase reporter driven by T cell activation pathway-dependent response elements); TIM-3 Target Cells (MHCII-positive human cell line). The TIM-3 Effector and Target Cells are provided in thaw-and-use format as cryopreserved cells that can be thawed, plated and used in an assay without the need for cell culture and propagation. When the two cell types are co-cultured, TIM-3 Target Cells provide low-level stimulation to the TIM-3 Effector Cells through the TCR. TIM-3 is engaged and activated by its ligands, including PS, which is present on both Effector and Target cells. The combined stimulation of the TCR and TIM-3 induces promoter-mediated luciferase activation and luminescence. Adding a TIM-3 blocking antibody prevents TIM-3 signaling and reduces promoter-mediated luminescence. The luminescent signal is quantified using the Bio-Glo-NL Luciferase Assay System and a standard luminometer such as the GloMax Discover System. In the TIM-3 Effector Cells, we find increased IL-2 reporter activity and secretion compared to the parental cell line. This elevated IL-2 reporter activity can be suppressed to parental levels by addition of TIM-3 antibody. The assay is therefore in agreement with the literature suggesting a stimulatory function for TIM-3. The TIM-3 Bioassay reflects the mechanism of action (MOA) of biologics designed to block TIM-3 signaling.