Zinc is a key micronutrient involved in many cellular processes and biological pathways including oxidative stress and inflammation. Therefore, we evaluated the effect of the intracellular bioavailability of Zn in bovine mammary epithelial alveolar cells (MacT) incubated at 0, 10, and 50 µM concentrations of Zn. Before transfection, MacT cells were cultivated in high glucose Dulbecco modified Eagle’s medium (DMEM) with sodium pyruvate and supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and Fungizone Antimycotic. The plasmid used in this study was the peZinCh-NB (Addgene) designed to detect intracellular Zn through a fluorescence resonance energy transfer (FRET) technology. Cells were seeded 24 h before transfection at 30,000 cells/well in a 96-well plate. Additional cells were seeded at 300,000 cells/well in a 6-well plate for gene expression analysis. Cells were transfected with the transfection reagent Lipofectamine 3000 at 0.3 μL/well and at 150 ng/well of plasmid in a reduced serum medium (OptiMEM) deprived of FBS. Transfected cells were treated for 24h in triplicates with 0, 10, and 50 μM Zn. An inverted fluorescent microscope for live imagining (EVOS FL Auto) equipped with a motorized scanning stage, and an environment-controlled chamber at 37°C and 5.0% of CO₂ was used to take 4 pictures/well at 4× magnification at 0, 12, and 24 h post-treatment. Quantification of Zn and cell viability were assessed using the CellProfiler software. Data were analyzed using the PROC MIXED of SAS. Overall intracellular availability of Zn increased (P < 0.01) in cells incubated with both 10 µM and 50 µM of Zn as early as 12 h post-treatment. At 12 and 24 h post-treatment, the greatest (P < 0.01) Zn intracellular bioavailability was observed with 50 µM of Zn, when compared with 10 µM of Zn and control. The cell viability at 24 h was similar (P ≥ 0.26) across treatments with 81.59, 80.92, and 75.06% for control, 10 µM and 50 µM of Zn, respectively. These preliminary data indicate that intracellular Zn can be detected via a fluorescent protein system in real-time in bovine cells. To expand on these effects, gene expression analysis will be performed.