A non-toxic and versatile protein salting-out DNA extraction method is here described for convenient and rapid extraction of nuclear DNA molecules from trypanosomatids. The procedure just involves four manipulations, does not require any organic solvent, and is performed in less than 1h in a single tube. DNA yields obtained were similar to those from commercial kits and phenol-chloroform procedures. Samples extracted by this method were suitable for PCR and subsequent analyses. The reduced manual labour involved was perceived as an important benefit in medical diagnosis routine use as well as for large-scale taxonomic and eco-epidemiological studies of trypanosomatids.