The intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires' disease, an acute form of pneumonia. After phagocytosis by lung macrophages, the bacteria replicate in a specialized vacuole termed the L. pneumophila containing vacuole (LCV) that resists fusion with late endocytic compartments and lysosomes and recruits vesicles that resembles rough endoplasmic reticulum. The bacterial Dot/Icm Type IV secretion system (T4SS) is required for LCV biogenesis and is responsible for the translocation of >250 bacterial effector proteins into host cells. Unlike other bacterial secretion systems, secretion of effectors by L. pneumophila does not occur in the absence of host cells. We hypothesize that the function of the Dot/Icm system requires active participation by the host cell. This study aims to identify host factors, which contribute to L. pneumophila entry into macrophages and/or effector translocation. This study will employ a genome-wide RNAi screen and a fluorescence reporter assay for effector translocation. The translocation assay measures FRET conversion of the fluorophore CCF2/AM, which quantifies the amount effector translocation. Here the assay was developed and optimized for use in high-throughput screens. A number of additional parameters were optimized for the genome-wide RNAi screen. These included choice of cell line, cell plating density, concentration and type of transfection reagent and duration of knockdown. The preferred parameters were then tested using trial siRNAs and knock-down efficiency was confirmed by western blot, microscopy and RT-PCR. The optimized parameters were successfully transferred and adapted from manual work to robotics at the genome screening facility. Therefore this study has established a system to perform genome-wide RNAi screening for the identification of host cell factors involved in L. pneumophila effector translocation and host cell invasion.