The cytogenetic identification is important for the characterization of an organism. In amphibians, the direct method is a technique of routine for chromosomal characterization, but is necessary for the sacrifice of the copies. The aim of this work is to develop the technique of lymphocyte cultures for the species Rhinella arenarum without sacrifice of specimen. Materials and Methods: Male specimens’ Rhinella arenarum were collected at San Luis; the blood sample was obtained by cardiac puncture. The mediums tested were: the culture media: RPMI 1690 with HEPES and Glutamine, MEM and F10; and different volumes of: phytohemagglutinin, penicillin-streptomycin, fetal bovine serum and colchicine. Results: For the standardization of the protocol we rely on cell culture techniques of fish and human. The following parameters were standardized: volume blood: volume of 100 μl; culture medium was chosen: the best results were observed with RPMI 1640; fetal bovine serum: he worked with a volume of 500 μl in the case of RPMI 1640 and F10, for MEM 1000 μl was used; phytohemagglutinin: it was observed that large volumes of this reagent agglutinated cells, and hence the greater number of metaphases was obtained with 10 μl; colchicine: best chromosome size was observed with 100 μl and an incubation time of 12 h. Conclusion: The standardized protocol is a simple and inexpensive technique that does not require equipment or facilities of high complexity and specimen are kept alive.