High throughput fluorescence polarization (FP) assays are described that offer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V1a and delta-opioid) and nonpeptide (beta1-adrenoceptor, 5-hydroxytryptamine3) receptors. Structure-activity relationships were investigated at beta1-receptors and were found to be consistent with radioligand binding assays. FP was shown to tolerate up to 5% DMSO with no loss in sensitivity or signal window. From a random set of 1,280 compounds, 1.9% were found to significantly interfere with FP measurement. If fluorescent or quenching compounds were eliminated (3% of all compounds), less than 0.4% of compounds were found to interfere with FP measurement. Assays could be run in 384-well plates with little loss of signal window or sensitivity compared to 96-well plate assays. New advances in FP measurement have therefore enabled FP to offer a high throughput alternative to radioligand binding for cell surface receptors.