Entry is the first and essential step in virus replication and is a target for therapeutic intervention. However, current knowledge on entry mechanism for the majority of viruses is poor, partly due to lack of a simple, sensitive and accurate entry assay that can be applied to diverse viruses. To overcome this obstacle, a novel contents-mixing-based virus entry assay is described that can be broadly applied to many enveloped viruses. By fusing firefly luciferase to the HIV Nef protein, luciferase was directly packaged into HIV particles pseudotyped with envelope proteins of diverse viruses including HIV, rabies and others. Upon cell entry, the luciferase-fusion protein was released into the cell cytoplasm, reacted with its substrates and was detected by light emission. The assay was validated by demonstrating its versatility in measuring virus entry. Entry was detected much more rapidly (in real-time) with higher sensitivity (a multiplicity of infection <0.1 gives a robust signal) and lower background (signal/noise ration >1000) than other comparable assays. In addition to its utility in studying virus entry mechanisms, the assay will aid in screening potential entry/fusion inhibitors and in diagnosis of virus infections.