Nonradioactive homogeneous assays are widely used to screen for inhibitors of biomolecular interactions. To ensure optimal sensitivity for the detection of competitive inhibitors, reagent concentrations should be fixed at or below the K(D) of the protein-protein interaction. Accurate measurement of K(D) during assay development is therefore critical. Although conventional methods work well with heterogeneous assays, they are generally unsatisfactory with homogeneous systems. Here the authors describe an alternative method to determine the K(D) of protein-protein interactions in homogeneous assays. The method uses a rearrangement of the Cheng-Prusoff equation: IC50= (([Ki]/K(D)) x [L]) + Ki. A competitive inhibitor is titrated into the ligand-receptor binding assay at a range of ligand concentrations and IC50 values are calculated. Plotting measured IC50 versus concentration of ligand gives a linear plot with y-intercept (Ki) and gradient (Ki/K(D)). K(D) is the affinity constant for the ligand-receptor interaction. Here the authors use homogeneous time-resolved fluorescence (HTRF) in 2 model systems (TRAIL/TRAIL receptor 4 and OX40 ligand/OX40 receptor) and demonstrate that measured K(D) values calculated using the linearized Cheng-Prusoff plot compare favorably with those from independent experiments. The advantages and limitations of the method are discussed.