Objective:  Survival of periodontal ligament cells is crucial issue after tooth replantation. To understand this matter, we introduced MTT assay, which can be used as a tool for measuring the viability of periodontal ligament cells from extracted rat molars.Materials and methods:  The maxillary molars of 4‐week‐old Sprague–Dawley white female rats were used. Ten teeth of each immediate, 1 h Viaspan and 1 h dry were processed for MTT evaluation. Another 10 teeth from each group were treated in the same manner as above, but were replanted into their original socket. After 2 weeks, the animals were killed and the prevalence of resorption pits was evaluated.Results:  In vivo MTT assay corresponded with the histological results of the resorption pits (P ≤ 0.05). The polarizing and optical microscopic findings were consistent with the in vivo MTT assay results.Conclusions:  In vivo MTT measurements were consistent with the histologic observations and suggest that the in vivo MTT assay could be a tool for evaluating the viability of periodontal ligament cells directly from the extracted root surface. The advantages are shorter analysis time compared with animal or cell culture experiments, easy manipulation, clear quantification and immediate identification of the vital cells.