An efficient and successful hit and lead discovery strategy is the basis of every low molecular weight (MW) drug reaching the market to address an unmet medical need. Until the early 1990s, the vast majority of new drugs were based on analogs of older drugs or natural products. Today, about 50% of the low MW new molecular entities receiving approval from the authorities are based on leads derived from screening and structural design. The paradigm of high-throughput screening has shifted toward assessing targeted libraries of smaller size, which have been selected based on physicochemical properties and/or by computational approaches (virtual screening). Biophysical methods play an increasing role in hit validation as the entry point for the hit-to-lead process.