Multigene delivery and subsequent cellular expression is emerging as a key technology for many applications in diverse research fields including, synthetic biology, structural analysis of multiprotein complexes, cellular reprogramming, genome editing and many others. We developed a baculovirus-mediated multigene expression system (MultiPrime), allowing simultaneous expression of several genes from a single virus in mammalian cells. MultiPrime can be used for the infection of primary cells or of cell lines that are difficult to transfect. Here, we show that it is possible to express up to five proteins with a modified baculovirus in both primary and established cells. We also created different intracellular biosensors which can be expressed simultaneously and allow to study receptor trafficking and signaling in single cells. MultiPrime-infected Zebrafish showed expression of all genes in vivo. MultiPrime virus was also used for CRISPR/Cas9-mediated generation of primary human knockin cells expressing fluorescently tagged HMGA1 from it native genomic locus. For this purpose, the virus contained a homology cassette, a CMV-driven Cas9 cassette, and a U6-drive cassette for gRNA expression. Taken together, baculovirus is an efficient vehicle to deliver multiple expression cassettes to mammalian cells and we show multiple applications of our system in cell biology.