In the current drug discovery paradigm, validated recombinant targets form the basis of in vitro high-throughput screening (HTS) assays. Isolated proteins cannot, however, be regarded as representative of complex biological systems; hence, cell-based systems can be employed to complement in vitro data, providing greater confidence in compound activity in an intact biological system. The scarcity of human material and the lack of proliferative capacity of primary cell cultures, combined with problems associated with the use of stem cells, mean that immortalized cell lines are generally used as a source of cells for HTS. While such cell lines have improved proliferative capacity, they often display aberrant genetic and functional characteristics. Consequently, interest has focused on creating new cell lines using defined molecular strategies that overcome senescence signals and telomere shortening. These strategies include expression of viral oncogenes and the catalytic subunit of telomerase (TERT) to generate immortalized cells. However, persistent proliferation induced by oncogenes can have undesirable effects, particularly on differentiation; hence, conditional immortalization approaches have been developed to provide more suitable cell models. The present review discusses the advantages of different technologies available for the generation of cell lines for use in in vitro biology, and describes a representative range of cell lines currently used in drug discovery.