The study of nuclear receptor activation by specific dietary compounds via gene reporter technology (GRT) is essential in nutrigenomics research. The GRT requires inserting into cells an artificial plasmid containing a promoter region with the response element for the nuclear receptor of interest and DNA coding for luciferase or a fluorescent protein. The main challenge of using GRT is the low efficiency and high variability of plasmid transfection; thus limiting the sensitivity and increasing intra-essay variability. To investigate the efficiency of transfection in immortalized (MacT) and primary mammary bovine (BMEC) cells we have tested several concentrations of transfection reagents in combination with several concentrations of a plasmid for the constitutively expressed enhanced green fluorescent protein (EGFP). Cells were seeded 24 h prior transfection at 30,000 cells/well in a 96-well plate and treated with a nuclear staining (NucBlue Live). The transfection reagents Lipofectamine 2000, Lipofectamine LTX, and TransIT-X2 were used at 0.2, 0.3, 0.4, or 0.5 μL/well with 50, 100, 200, or 300 ng/well of EGFP. Using a robotic inverted fluorescent microscope for live imaging (Leica DMI6000B), 2 pictures/well were taken every hour for 30 h post-transfection. Cell number, viability, and quantification of transfection efficiency were assessed using the CellProfiler sotfware. Data were analyzed using GLIMMIX of SAS. EGFP protein was visualized as early as 4 h after transfection and plateau expression was observed >7 h post-transfection. We observed high variability in transfection efficiency ranging from 1 to 30%. Transfection efficiency was best using Lipofectamine 2000 in BMEC and Lipofectamine LTX in MacT. In general we observed that high dose of transfection reagent and plasmid provided best results; however, high dose of transfection reagent tend also to kill the cells. Overall, our data confirmed the large intra- and inter-cells variation in transfection efficiency and prompt for a more precise approach to obtain high reliable data for nutrigenomics studies in dairy cows.