Phlebotomine sand flies are blood-feeding insects that transmit Leishmania parasites that cause various forms of cutaneous and visceral leishmaniasis and sand fly fever viruses (Phlebovirus; Bunyaviridae) in humans. Sand flies pose a significant threat to US military personnel deployed to Leishmania-endemic and sand fly fever endemic regions which include Europe, the Mediterranean basin, Middle East, Central Asia, Southwest Asia, and Africa. A research project supported by the Department of Defense Deployed Warfighter Protection Program was initiated to evaluate the susceptibility of 2 Old World sand fly species, Phlebotomus papatasi and P duboscqi, to a number of commonly used pyrethroid and organophosphate insecticides. A new glass vial bioassay technique based on the CDC bottle assay was developed for this study. The exposure time-mortality relationship at a given insecticide concentration was determined for each insecticide, and their relative toxicity against the 2 sand fly species was ranked based on bioassay results. This study validated the new bioassay technique and also generated baseline insecticide susceptibility data to inform future insecticide resistance monitoring work.