Here we describe the efficient production of native RSPs of the four DV serotypes by using a codon optimization strategy. Codons of the DV prME gene were replaced with those preferentially used in mammalian cells. Optimization was first applied to the DV1 prME gene. We found that this experimental approach could efficiently increase the intracellular expression level of the E glycoprotein in human cells and therefore enhance the production of DV1 RSPs. We have established a DV1 RSPs producing stable cell line (HeLa-prME). Our data by immunofluorescence and electron microscopy show that most of E protein co-localizes with markers of the ER, where RSPs accumulate. Biochemical analysis of secreted RSPs demonstrates that they contain E homodimers and M, indicating that RSPs have trafficked through the secretory pathway and that maturation process has occurred. Using the HeLa-prME cell line, we have developed a semi-quantitative assay to screen a 122 genes cellular membrane trafficking siRNA library and identified genes that may be involved in the secretion of DV. From our screen, 23 genes show a significant reduction in DV1 RSPs secretion whereas for 22 genes RSPs levels were increased in cell supernatants. These data provide a proof of concept that RSPs of DVs and the producing cell line are safe tools that can be used in the study of viral egress. Finally, the optimization strategy was applied to DV2, DV3 and DV4 prME, and RSPs for all four DV serotypes were efficiently produced. These native RSPs are, therefore, a safe mimicry of virions that can be used to study viral and cellular requirements for virus assembly and egress.