Live cells, tissue or cell homogenate containing the receptor of interest is incubated together with a radioligand for the receptor and a range of concentrations of the test (competing) drug. Typically up to 11 concentrations of the test drug are evaluated over a three log unit range and with triplicate (or quadruplicate) replicates at each concentration.

After equilibrium has been reached, receptor-bound radioligand is separated from non-bound radioligand using either.

• Filter-based Separation
• Scintillation Proximity Assay (SPA)

The filters or SPA plate are counted for radioactivity to determine the level of receptor-bound radioligand and the results are plotted to obtain the IC₅₀ and K_i values for the drug-receptor interaction.