Cell-based assays for the inhibition of viral infections most commonly couple a positive signal (e.g., an increase in fluorescence) to the infection itself and not to its inhibition. When performing drug screens, compounds decreasing the signal are therefore considered as putative inhibitors. However, this approach can cause the selection of many false positives, since, for example, both killing of the host cell and inhibiting viral cell-entry results in the same signal. Using a model system based on murine leukemia virus (MLV) particles pseudotyped with the G-protein of vesicular stomatitis virus (VSV-G), we have developed generic assays coupling a positive readout to the inhibition of viral transduction. Consequently, the system favors drug candidates (and concentrations thereof) that do not harm human cells and significantly decreases the probability for selecting false positives. The assay allows Z-factors of approximately 0.9, takes cytotoxic side effects into account and could in theory be adapted for high-throughput screening of inhibitors against further viral species.