African swine fever (ASF) is a devastating disease of pigs causing a global pandemic with mortality rates reaching 100%. ASF vaccine development has been extremely difficult due to the complexity of the ASF virus (ASFV), which has a tropism for cells within the mononuclear phagocyte system, eg, macrophages. Live attenuated vaccines (LAVs) are currently the most promising options and are approved for use in a small number of countries. However, there is a recognised need for alternate “non-live” next generation vaccines to address the safety concerns that LAVs pose. Disabled infectious single cycle (DISC) viruses offer a safer alternative to LAVs. DISC vaccines are developed by deleting replication essential gene(s) that limit the infection within the host cells to only one cycle, while allowing expression of viral proteins required for the generation of robust immunity. To identify essential ASFV gene(s), we are developing a tetracycline (Tet) dependent expression system to achieve regulatable expression of target viral genes. For this, a Tet operator is integrated with the target genes in the ASFV genome. The recombinant virus is complemented by a Tet repressor expressing cell line that is susceptible to ASFV. When the Tet repressor cells are infected with recombinant ASFVs encoding a Tet operator, a Tet-dependent regulation will function to suppress the expression of the target ASFV genes. This system can thereby be used to test the essential function of the affected gene in the viral replication cycle. We will employ this approach to assess a panel of candidate ASFV genes to interrogate their roles in the viral replication cycle and to select genes that can be incorporated in the development of a next generation ASFV DISC vaccine. This paper will present progress made towards the identification of essential viral genes of a major animal pathogen.