Stem cells from human exfoliated deciduous teeth are a population of highly proliferative postnatal stem cells and have been characterized as multipotent stem cells. In this study we developed a fast and sensitive method for stem cells derived from human exfoliated deciduous teeth count, using a luminescent viability assay. We isolated stem cells from normal exfoliated deciduous teeth using collagenase/dispase digestions solutions. Separated stem cells were placed in opaque-walled multiwall plates in culture alpha Modified Eagle's Medium. For dental pulp stem cells quantitation we used a simple method for determining the number of viable cells based on ATP concentration. Cells attached to the bottom of the multiwall plates were counted with the luminescent assay and were cultured for mesenchymal markers expression. Moreover cells attached to the bottom of the multiwall plates were directed toward the osteogenic, adipogenic, lineages at the respective passages. Flow cytometry was used for immunophenotyping of cultured dental stem cells from exfoliated deciduous teeth. Cells that were counted with the luminescent assay, after culture formed fibroblastic morphology and were expressed the mesenchymal stem cell markers CD29, CD105, CD146, CD44. There was a correlation between the number of cells plated for culture and the number of mesenchymal stem cells after culture. Osteogenic and adipogenic differentiation of the cells counted with the luminescent assay was performed. The luminescent signal of viable mesenchymal dental stem cells isolated from dental pulp of exfoliated teeth represents an ideal method for mesenchymal stem cells count before culturing.