Embryonic stem (ES) cells, combining self-renewal ability with wide range tissue-specific cell differentiation, represent one of the most powerful model systems in basic research, drug discovery and biomedical applications. In the field of drug development, ES cells are instrumental in high-throughput/content screening (HTS/HCS) for the evaluation of large compound libraries to test biological activity and toxic properties. Since it is a high priority to test new compounds in vitro, before starting animal and human treatments, there is an increasing demand for new in vitro models that can be used in HTS/HCS to facilitate drug development. In order to achieve this objective, several methods for ES cell self-renewal or differentiation have been evaluated to assess their compatibility with HTS/HCS. This review describes protocols used to screen molecules able to maintain self-renewal or to induce differentiation in ectodermal, mesodermal, endodermal, and their derivative cell lines.