The use of high-throughput screening for early stage drug discovery imposes several constraints on the format of assays for therapeutic targets of interest. Homogeneous cell-free assays based on energy transfer, fluorescence polarization spectroscopy or fluorescence correlation spectroscopy provide the sensitivity, ease, speed and resistance to interference from test compounds needed to function in a high-throughput screening mode. Similarly, novel cell-based assays are now being adapted for high-throughput screening, providing for in situ analysis of a variety of biological targets. Finally, recent advances in assay miniaturization mark a transition to ultra high-throughput screening, ensuring that identification of lead compounds will not be the rate-limiting step in finding new drugs.