Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.