F694-1532 inhibits viral replication by an as yet unknown mechanism. cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 D-γ-Glutamyl-D-glutamic acid exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their mechanism of action and any possible deleterious effects on host cellular biology. Author Summary Rift Valley fever (RVF) is an arthropod-borne viral zoonosis that occurs in large parts of sub-Saharan and North Africa and in 2000 emerged outside the African continent for the first time, raising concerns that it could further expand its geographical range. The disease in humans can result in encephalitis or hemorrhagic fever and in ruminants often results in abortion in pregnant females. Due to the lack of a licensed and D-γ-Glutamyl-D-glutamic acid commercially available vaccine, efforts to discover effective Col4a4 antiviral drugs are underway. Drug discovery using high content image-based screening is an effective tool that has been successfully used to identify new drugs. In this study, we developed an image-based assay to identify compounds active against RVF virus and other highly pathogenic human viruses. We demonstrated the usefulness of our image-based high content assay in identifying potential RVF antivirals by screening a small subset of chemical compounds for inhibition of RVF virus in a human cell line (HeLa) and partially characterized their mechanism of action within infected cells. The methods we developed in this study will be useful in discovering new effective drugs to combat Rift Valley fever. Introduction Many RNA viruses are highly pathogenic to humans and can cause hemorrhagic fever and/or encephalitis. Among these, Rift Valley fever virus (RVFV), a member of the genus (family family, causes severe encephalitis in horses and humans (reviewed in ). RVFV, as with other highly pathogenic RNA viruses, including EBOV, MARV, VEEV and LASV, cause severe disease in many developing countries that already suffer from fragile economies and health care infrastructures. There is currently no U.S. D-γ-Glutamyl-D-glutamic acid Food and Drug Administration (FDA) approved therapeutic or prophylactic treatments for D-γ-Glutamyl-D-glutamic acid any of these agents, thus there is an urgent need for research to develop effective new drugs and vaccines to combat these diseases. Recent advancements in high content image (HCI)-based screening (HCS) technologies have contributed greatly to increasing the efficiency of the drug discovery process. HCS utilizes automated high-speed, high-resolution microscopy and image analysis to measure morphological changes in the cells in a quantitative and high-throughput manner . Most importantly, HCI-based analysis enables simultaneous measurement of multiple features of cellular biology that are relevant to therapeutic and cytotoxic characteristics of potential antiviral compounds. As a result, HCI-based analyses not only allows for rapid screening of compounds, but can provide early insights into their cytotoxicity and mode of action, thereby facilitating the decision-making processes that govern the progression from a candidate compound to a successful antiviral drug. RVFV is an enveloped spherical virus with containing a has a tri-segmented, single-stranded RNA genome, which encodes for the RNA-dependent RNA D-γ-Glutamyl-D-glutamic acid polymerase (RdRp), envelope.