Animal cell lines have become very popular substrates for the production of vaccines and biopharmaceuticals. Characterization of candidate production cell lines is central to ensure product safety and maintenance of consistency in the manufacture of biologicals. Nested PCR and isoenzyme analysis have been used widely to prove the identity and purity of various cell lines and primary cells individually and also after deliberate cross-contamination. The nested PCR based on the Cytochrome b (Cyt b) gene of mitochondrial DNA (Mt DNA) was found to be more sensitive than isoenzyme analysis in detecting low levels of contaminants (as low as 1%). Interestingly, competition between different co-cultured cell lines has shown in one case that cross-contamination need not always results in a mixed cell population. The nested PCR technique for the Cyt b gene described in this study appears to be a potential replacement for isoenzyme analysis and here we demonstrate the PCR method used is sensitive and reliable for cell line authentication in a simple, rapid and reliable format to help assure the authenticity of cell substrates for the production of safe vaccines and biopharmaceuticals.