Cigarette smoke condensate (CSC) is known to inhibit cell communication in a dose-dependent manner. We developed and evaluated a new high throughput GJIC assay for reproducible dose response, accuracy, and precision. An automated fluorescent microscope (ArraySCAN®, cellomics), the rat liver epithelial cell line WB-F344, and CSC from the 2R4F Kentucky reference cigarette and two single-tobacco cigarettes (i.e., bright and burley) were utilized in the evaluation. Experimental: cells were stained with the fluorescent dye calcein. Stained cells were pipetted to unstained cells of the same type and incubated for 3 h with the test substance or solvent control (0.5% DMSO). After establishing the connexin channels, the transfer of the fluorescent dye to neighboring cells within 3 h was determined and expressed as “% stained cells” as a measure of gap-junctional intercellular communication. Phorbol-12-myristate-13-acetate (TPA), a well-known inhibitor of GJIC, was used as a control. Results: the assay showed a reproducible dose response. The repeatability and reproducibility for the 2R4F were 3.7% and 6.9%, respectively; the minimal detectable difference was 5.7 µg/ml. The EC50 value for TPA was 0.34 ng/ml, which is in the range reported in the literature for other GJIC assay designs. The ec50 values for CSC from the three cigarette types were 45 µg/ml for bright, 51 µg/ml for the 2R4F, which is a mixture of bright and burley tobacco, and 58 µg/ml for burley, which had a shallower slope than the other two cigarette types. Conclusion: this cell-based assay can determine the GJIC-inhibitory activity of CSC, and meets ISO criteria for precision and accuracy. The assay can also discriminate between different CSCS, as reflected by the ec50 values for the three cigarette types. In conclusion, this screening assay is an adequate tool to determine GJIC, which has been proposed to play a role in tumor promotion.