Co-stimulatory checkpoint receptor signaling assays measure activation of a member of the TNFR family of receptors, which activates the non-canonical NF-kB pathway through NF-kB inducing kinase (NIK). The cell line is engineered to express NIK labeled with enzyme donor (ED). NIK protein levels inside the cell are tightly maintained at low levels by the proteasome, and increase in response to pathway activation. Addition of exogenous Enzyme Acceptor (EA) forces complementation of the ED and EA enzyme fragments resulting in the formation of a functional β-gal enzyme that hydrolyzes substrate to generate a chemiluminescent signal. The assays may rely on either endogenous receptors or exogenously expressed target receptors.