Quantification of Leishmania parasites in the sand fly digestive tract is important for evaluation of vector competence. We compared quantitative polymerase chain reaction (Q-PCR) with two "traditional" methods, estimation in situ and direct counting with the aid of a hemocytometer, to evaluate their usefulness in different parasite-vector combinations. Phlebotomus duboscqi Neveu-Lemarie and Phlebotomus arabicus Theodor sand flies were infected with Leishmania major and Leishmania infantum, respectively, and different approaches were compared to determine the intensity of Leishmania infections before and after defecation of the bloodmeal (on days 2 and 8 postinfection, respectively). Estimation of parasite numbers in situ is only a semiquantitative method, but it is quick and provides data about localization of infection. We recommend this technique for low-intensity infections after the bloodmeal is passed. Counting in a hemocytometer is a suitable technique for heavily infected sand flies or for quantification of Leishmania within the bloodmeal. Because of its relatively high cut-off (60 parasites per gut), it is not useful for low-intensity infection soon after defecation when parasites are attached to midgut. The most accurate approach for parasite quantification in any type of sand fly infection is Q-PCR. This method is also highly sensitive and can detect one parasite per gut. Localization of a Leishmania infection in the sand fly midgut is a parameter equally important to parasite numbers. Therefore, to get full information about the Leishmania development in sand flies, we propose to combine various techniques. Both Q-PCR and counting with a hemocytometer always should be preceded by in situ examination under the microscope to assess the localization of the infection.