Objective: Various studies have shown that the root surface condition may play an important role in wound healing. Root surface demineralization has been shown to promote the establishment of new connective tissue attachment. Various agents, including citric acid, ethylenediaminetetraacetic acid (EDTA), and tetracycline, have been used to try to achieve a root surface that is biocompatible with the adjacent periodontal cells. Traditional in vitro studies have used periodontal ligament fibroblasts and scanning electron microscopic studies to check the efficacy of these root-conditioning agents. The objective of this study was to assess the efficacy of trypan blue to evaluate fibroblast attachment in an in vitro model using V79 fibroblasts (embryonic Chinese hamster lung fibroblasts) on root specimens treated by citric acid, EDTA, and tetracycline. Method and materials: Citric acid-, EDTA-, and tetracycline-treated root specimens were placed in cultures of V79 cells and in human periodontal ligament cells, which acted as a control. The treated root specimens were removed from the cultures after 4 weeks and immediately treated with 1% trypan blue. Trypan blue was selected because it can stain only nonvital tissues. Results: The root specimens placed in V79 and human periodontal ligament cultures showed unstained areas, indicating the presence of vital cells, in contrast to the stained areas, which represented the areas devoid of cells. Conclusion: This in vitro screening model based on the trypan blue exclusion principle may be used for immediate evaluation of the efficacy of various root-conditioning agents. V79 cells may be used as an alternative to human periodontal ligament fibroblasts in the evaluation of the efficacy of root conditioning agents. Keywords: periodontal ligament fibroblasts, root conditioning, trypan blue exclusion principle, trypan blue stain, V79 cells