Overexpression of the gene of interest is commonly used in applied and basic biosciences to study the in-depth functional importance of protein. A deeper knowledge of protein interaction enhances our understanding of biological complexity. However, there is a scarcity of suitable and robust methods to investigate the diverse functions of the 3′ untranslated region (3′UTR), a specific region located at the 3′ end of the mRNA, as identifying the interacting partners of 3′UTR is challenging. The Flp-In model system enables integration and expression of the gene of interest at the specific genomic locus of mammalian cells. In this study, we used a Flp-In system to construct a double stable cell line that simultaneously expresses chimeric MS2 bacteriophage coat protein and 3′UTR. Double-stable cell line expresses a gene of interest over an extended period of time in a highly reproducible manner in different experimental conditions. We present the construction of required vectors and provide a complete procedure for transfection and validation of integration of the desired genes at a genomic locus and their expression at the RNA and protein level. This model system provides a platform for deciphering the role of 3′UTR, providing deeper insight into its implications in health and diseases.