The ability of transposable elements to mobilize across genomes and affect the expression of genes makes them exceptional tools for genetic manipulation methodologies. Several transposon-based systems have been modified and incorporated into shuttle mutagenesis approaches in a variety of organisms. We have found that the Mos1 element, a DNA transposon from Drosophila mauritiana, is suitable and readily adaptable to a variety of strategies to the study of trypanosomatid parasitic protozoa. Trypanosomatids are the causative agents of a wide range of neglected diseases in underdeveloped regions of the globe. In this chapter we describe the basic elements and the available protocols for the in vitro use of Mos1 derivatives in the protozoan parasite Leishmania.