The AlphaLISA® mouse PD-1/PD-L1 binding assay enables development and characterization of small and large molecule anti-mouse PD-1 or PD-L1 reagents for in vitro and in vivo studies, testing of other putative binding ligands such as PD-L2 or characterization of ligand subregions for their ability to block binding, and determination of mouse cross-reactivity of anti-human PD-1 or PD-L1 reagents using a homogeneous AlphaLISA assay (no wash steps).