Th e use of cell culture in biomedical science has been constantly enhanced and the use of certifi ed and microrganismsfree cell lines is indispensable. Due to the value and the diffi culty of acquiring a commercially available kit (Authentikit TM system) for our routine in addition with the purpose of monitoring and ensuring quality of provided cell lines, and also contributing with cell characterization, the Núcleo de Cultura de Células do Instituto Adolfo Lutz (NCC-AL) padronized two techniques to identify the animal species from which the cells were originated: isoenzyme electrophoresis and karyotype. Cell extracts from 14 diff erent cell lines were run under horizontal electrophoresis in agarose gel and lactate dehydrogenase isoenzyme profi le was revealed using tetrazolium salt. Th e number of bands and the migration distances between bands of cell lines were similar to the literature, but small adjustments were needed to assist in the visualization of the bands that were not visible. Cell fl asks for karyotype were incubated with colcemid solution and modal values of each cell line were calculated. Comparing all the results obtained with those reported in the literature, we conclude that both methods are promising and will allow better control of the services provided by the laboratory, not to mention guaranteeing authenticated cell lines for research and diagnostics.