Rodent transgenic parasites are useful tools for the preclinical evaluation of malaria vaccines. Over the last decade, several studies have reported the development of transgenic rodent parasites expressing P. falciparum antigens for the assessment of vaccine-induced immune responses, which traditionally have been limited to in vitro assays. However, the genetic manipulation of rodent Plasmodium species can have detrimental effects on the parasite’s infectivity and development. In this chapter, we present a few guidelines for designing transfection plasmids, which should improve transfection efficiency and facilitate the generation of functional transgenic parasite strains. In addition, we provide a transfection protocol for the development of transgenic P. berghei parasites as well as practical methods to assess the viability and infectivity of these newly generated strains throughout different stages of their life cycle. These techniques should allow researchers to develop novel rodent malaria parasites expressing antigens from human malaria species and to determine whether these transgenic strains are fully infectious and thus represent stringent platforms for the in vivo evaluation of malaria vaccine candidates.