In the development of novel substrates used for production of human vaccines there has been significant progress made in recent years. Emerging and re-emerging infectious diseases like the recent porcine Influenza A virus (H1N1) pandemic necessitated the availability of unprecedented amounts of vaccines. In addition, the high demand for vaccines in the industrialised countries has also been paralleled by a steep increase in demand in developing countries. The manufacturing capability for viral vaccines produced in embryonated hen eggs and conventional/classical cell substrates, such as chicken embryo fibroblasts, has now reached its capacity limit. This constraint may be overcome by utilising other recognised cell substrates such as Madin Darby Canine Kidney (MDCK) (dog origin), Chinese Hamster Ovary (CHO) (hamster cells) or Vero cells (monkey origin) or as an alternative, introduce new cell substrates of human or avian origin. Using new cell substrates may prove to be a highly replication-proficient way of producing live viral vaccines such as Influenza A viruses. Despite some advantages, cell substrates may pose a small residual risk to humans since some of them are known to be tumourigenic in immunosuppressed animals. However, this residual risk should be considered acceptable by regulators. Safety testing requirements for cell substrates used in the manufacture of vaccines is mandated by published guidance from organisations such as World Health Organization (WHO), United States Food and Drug Administration (FDA), European Medicines Agency (EMA) and International Conferences on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH) as well as requirements laid down in compendial monographs (Ph. Eur. and USP). This paper considers the guidance contained in these regulatory documents. In addition, the safety challenges and almost arbitrary risk-based classification of cell substrates used in the production of human vaccines together with compliance to GCCP (Good Cell Culture Practice) are discussed. Even though there has been tremendous progress in the last few years, reflected mainly by revisions and updates to regulatory guidance documents, there still is still no consensus between regulators nor significant harmonisation of the guidance documents or monographs.