The present work evaluates sampling protocols, storage procedures, and DNA purification methods for Leishmania spp. detection and quantification in different biological samples. The efficiency of three preservation solutions, a phosphate buffer solution, an ethylenediaminetetraacetic acid (EDTA) buffer solution, and 70% ethanol, was compared in combination with three DNA extraction protocols: a commercial silica column kit, salting-out protein precipitation, and organic extraction with phenol-chloroform. Tissue samples from BALB/c mice experimentally infected with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, or Leishmania (Leishmania) infantum were stored in the three preservation solutions and subsequently subjected to the three different DNA extraction methods. The extracted DNA was then used in real-time polymerase chain reaction (PCR) assays for the detection and quantification of parasite ribosomal small subunit DNA targets as well as mammalian glyceraldehyde-3-phosphate dehydrogenase (gapdh) targets. The results of the optimized protocols showed that the DNA extraction method did not influence test quality, but DNA from samples preserved with the EDTA buffer solution produced higher amounts of target amplicons. Based on these results, we concluded that samples from suspected cases of leishmaniasis for submission to molecular diagnostic procedures should be preferentially preserved in EDTA, followed by any one of the DNA purification methods evaluated.