ELISA is one of the most widely used methods in immunology, molecular biology, and medical diagnostics. It enables the detection and quantification of specific proteins, antibodies, antigens, or hormones in biological samples such as blood serum, urine, or cerebrospinal fluid. ELISA is highly sensitive and specific, which makes it extremely useful in various fields of scientific research and clinical diagnosis. In recent years, this method has gained importance in the context of research on immune checkpoint inhibitors. Immune checkpoint inhibitors, such as antibodies, peptides, or small-molecule compounds that block receptor/ligand complex formation, have become modern therapeutic tools in immuno-oncology. Therefore, more and more research is being conducted in this field. ELISA is a relatively cheap and rapid method that can be effectively used to verify the initial inhibitory potential of compounds before more expensive and much more sophisticated cell-based assays are performed. In this protocol, we described how the inhibitory properties of compounds toward immune checkpoint complex formation can be investigated using ELISA and provided a detailed procedure that was used to evaluate the inhibitory properties of peptides toward the BTLA/HVEM complex formation.