Over the years, many different high throughput screening technologies and subsequently follow-up methodologies have been developed. All of these can be categorized, for example according to measurement of analyte classes, assay mechanisms, readout principles, or screening of drug target classes. When categorized according to drug target class, assay formats can be subdivided into early hit stage assays (usually ligand-binding based) that are often straightforward and robust up to analysis of final cellular effects exerted by ligands. The latter do not only provide higher content data but also represent anticipated effects in the body more closely. These assay formats, however, are often also elaborate, non-robust, and very time consuming to conduct but become pivotal when going from the hit-to-lead discovery stage. Looking at the hit-to-lead process, new assay possibilities in terms of measured effects and readout principles are continuously reaching the screening arena. Furthermore, at the (early-) lead discovery stage other targets also have to be evaluated to study e.g. target selectivity and ADME(T). This review discusses many of the different assay formats used in the hit-to-lead discovery phase sorted by their use for screening major drug target classes for small molecule drug discovery. The receptor drug targets in this review are subdivided into GPCRs, nuclear receptors and ion channels, while for the enzyme drug targets the important protein kinases, proteases and the drug metabolism enzymes (CYPs; mainly important for drug metabolism and drug-drug interactions) are discussed in more detail. For every drug target, different assay formats used to analyze ligand mediated effects at specific points in the drug targets respective signal transduction cascade(s) are looked at. More specifically, assay methodologies for ligand binding and second messenger formation up to signal transduction cascades and analysis of eventual (cell based) effects are described. Furthermore, special attention is paid to less traditional (non-platereader based) state-of-the-art screening approaches such as LC-MS, microarrays, microfluidics and sensor based technologies.