Crotamine is a natural cationic polypeptide isolated from the venom of South American rattlesnake (Crotalus durissus terrificus) capable of forming complexes with DNA molecules by means of electrostatic interactions and translocating biological membranes. With such properties, crotamine can act as a transfection agent for several cell types but mainly in a stage of proliferation. However, to improve the biological use of this peptide, it is interesting to better characterize the formation and stability of crotamina-DNA complex. Thus, the present study aimed to analyze the stoichiometry, formation kinetics and protease stability of crotamina-EGFP complex using high sensitive fluorescent methodology, based on fluorophore exclusion assay. The best DNA:crotamine mass proportion for complex formation ranges between 1:10 to 1:40 as mass ratio. In 1:10 group, crotamine was able to complex about 75% of the available DNA as fast as in 20 seconds, and after 10 min of reaction crotamine reached its maximum complexation capacity, being able to complex 80% of total amount of DNA available. Under these conditions, we have determined that 1μg of crotamine can complex 80 μg of circular plasmid DNA (~4700 bp). Crotamine-DNA complex was resistant to proteinase K degradation even after twelve hours of enzymatic incubation. Additionally, the complex was sufficiently stable to trypsin digestion for a long incubation time: more than 73% of crotamine-DNA condensates maintained the integrity after 60 min, whereas 55% was intact after 180 min. Crotamine-DNA complexes were resistant to physiological temperatures, as well as stable for 15 days at 4°C and for two months when stored at -20°C. These results lead us to think that, in the best conditions of preparation proposed here, crotamine-DNA condensates can be used in an efficient and standardized manner as a potential agent of transfection in a transgenesis programme.