Leishmania protozoan parasites are the causative agent of leishmaniasis, a disease which is endemic in Asia, Africa, the Americas, and Mediterranean regions. There are two main forms of leishmaniasis, visceral leishmaniasis which is a lethal condition and cutaneous leishmaniasis which causes lesions on the skin which heal on their own. Cutaneous leishmaniasis is caused by several species of Leishmania, the most common being L. major. Leishmania parasites are transmitted to humans through the bite of the sandfly, and once in the skin the parasites will elicit an innate immune response and will be phagocytosed into macrophages. The parasites will then transform and replicate inside of the macrophages. Recent studies have shown that the parasite load of L. major varied over 24 hours within macrophages and there was a rhythmic expression of cytokines. This rhythmic expression suggests that the circadian clock within immune cells controls the magnitude of infection. It is not known how the macrophage circadian rhythms affect the Leishmania infection.Therefore, our objective is to study how the macrophage circadian rhythms have an impact on Leishmania major infection. Imaging flow cytometry indicated that our infection protocol leads to 49.5% of bone marrow derived macrophages internalizing CFSE labelled parasites 6 hours after infection. Within the same population of macrophages, we observed different rates of infection with highly infected and low infected cells. However, our qPCR data analyzing activation of cytokines revealed that there was no activation of IL-6, TNFa or IL-1b within infected macrophages. No activation of cytokines suggests that our parasites are not eliciting an immune response within the macrophages, we may not be infecting with the correct ratio of parasites, or our parasites may not be highly virulent. In conclusion, the infection protocol we have developed works as macrophages are internalizing parasites, however the Leishmania are not activating an immune response within macrophages.