Ensuring the biosafety and quality of human vaccines is achieved through a multi-tiered approach that examines several factors to establish product safety and manufacturing consistency. The manufacture of vaccines can be both complex and challenging. There are a number of key process goals which are maintained to achieve scalable processes to ensure reproducibility of product. We provide an analysis of the current regulatory requirements for cell substrate testing and characterization of vaccines . Current testing methods are reviewed and presented to ensure understanding of methods to provide assurance of viral safety. The use of state of the art techniques to improve and expand existing testing processes to examine process product related impurities, identity and viral safety provides a level of both safety and quality assurance which address current regulatory expectations will also be presented.