Insect cells have shown a high versatility to produce multiple recombinant products. The ease of culture, low contamination risk with human pathogens and high expression capacity makes an attractive platform to generate virus-like particles (VLPs). The baculovirus expression vector system (BEVS) has been frequently used to produce these complex nanoparticles. However, the BEVS entails several difficulties in the downstream phase as well as undesirable side-effects due to the expression of baculovirus-derived proteins. In this work, we developed a baculovirus-free system based on polyethylenimine (PEI)-mediated transient gene expression (TGE) of Sf9 cells. An exhaustive study of DNA:PEI polyplex formation was performed and the optimal TGE conditions were determined by the combination of Design of Experiments (DoE) and desirability functions. The TGE approach was successfully applied to produce three model recombinant products with different structural complexities, including eGFP, hSEAP and HIV-1 Gag VLPs. Cell membrane co-localization with the Gag polyprotein was detected by fluorescence microscopy, whereas nanoparticle tracking analysis and flow virometry were applied as high-throughput techniques to monitor the VLP production process. Analysis of VLP production revealed that 48 h after transfection were optimal for VLP harvesting since the ratio of VLPs to extracellular vesicles was the highest. In these conditions, a maximum of 1.9 ± 0.8·109 VLP/mL was achieved, representing a 2.8-fold increase compared to the initial transfection condition. In conclusion, the TGE approach proposed in this study provides a baculovirus-free platform to rapidly produce VLPs and potentially other recombinant products in insect cells.