Combinatorial chemistry technology has enabled the synthesis of large, targeted libraries consisting of a much higher ratio of pharmacologically active compounds than traditional compound archives. This has resulted in a higher number of compounds requiring follow-up characterisation in full 50% inhibitory concentration (IC50) curves after the preliminary screen. The aim of this study was to develop a new assay format and analysis protocol for IC50 determination from the minimum number of data points so that more information could be derived from a primary inhibition screen. Data points from existing 10-point IC50 curves were used retrospectively to test the accuracy of IC50 predictions derived from just one or two compound concentrations. Regression analysis showed that both methods were useful, although, as expected, two compound concentrations gave more accurate IC50 predictions. A final experimental data set comparing IC50 values derived from a two- and 10- point assay format gave highly comparable data (r2 = 0.89). This study shows that it is possible to generate IC50 values from an appropriately designed primary inhibition screen using two compound concentrations, reducing the requirement for follow-up IC50 determinations.