Immune checkpoint blockade is an important cancer immunotherapy strategy that enhances the immune system’s ability to recognize and destroy tumor cells. The PD-1/PD-L1 signaling pathway is a key immune checkpoint that suppresses T-cell activity and allows cancer cells to evade immune surveillance. Blocking the interaction between PD-1 and PD-L1 can restore immune function and improve anti-tumor responses. The PD-1/PD-L1 Inhibitor Binding Assay is a TR-FRET–based assay designed to quantitatively measure the interaction between PD-1 and PD-L1. In this assay, binding of PD-1 to PD-L1 generates a fluorescence resonance energy transfer (FRET) signal that can be detected by measuring fluorescence emission at 665 nm and 620 nm. Compounds or antibodies that inhibit PD-1/PD-L1 binding reduce the TR-FRET signal, enabling evaluation of potential immune checkpoint inhibitors for cancer research and drug discovery applications.