Herpes simplex virus type 1 is one of the most widespread human viruses. The HSV-1 infection causes persistent infection which can be divided into two phases: a productive and latent phase. Latent phase is characterized as non-productive phase placed in neurons. Viral reactivation often occurs as a result of stress or immunocompromised state of the host. During viral reactivation, HSV-1 goes to a productive phase which is placed in epithelial cells and is characterized by viral replication. HSV-1 has developed a range of mechanisms by which it modulates host defense and provides efficient viral replication. One of the crucial proteins included in modulating host defense is ICP34.5 which is also the central protein of this study. ICP34.5 is a multifunctional protein and important neurovirulence factor. It interacts with many cellular proteins such as NOP53, PP1, PKR, etc. and ensures efficient viral replication. The purpose of this study is to determine experimentally the localization of ICP34.5 and link to its functions. The plasmid pcDNA 3xFLAG 34.5 with viral gene γ34.5 was used to express the protein of interest, and it contains three sequences of a FLAG tag, HSV-1 γ34.5 gene, and promoter from cytomegalovirus. pcDNA 3xFLAG 34.5 was transfected into human retinal epithelial cells (RPE-1). The localization of the ICP34.5 protein was analyzed using fluorescent microscopy. The results show that ICP34,5 is mostly expressed in the nucleus of transfected cells, which indicates its functions in the nucleus. It was also mildly expressed in the cell cytoplasm. In addition, we have observed that the amount of transfected plasmid shows a significant influence on the interpretation of the protein localization.