In general, pulpotomy is evaluated by pathological examination of sacrificed animals at each observing stage. Not only lots of small animals have to devote itself to experimentation, but also the result of research is lacking about the serial observation of an individual experimental animal. The aim of this study was to compare the relative effectiveness of the calcium hydroxide, formocresol and CO₂ laser using the R_mCT^®. Twenty male Wistar rats, 8 weeks of age, were used and imaged in present study. They were serially observed using the R_mCT^® on days 0, 3, 10, 30, 50 after pulpotomy, followed pathohistological examination. The R_mCT^® images revealed that the pulp capping agents were partly absorbed and a hard tissue-like was observed immediately beneath it, especially in the CO₂ laser group. Pathohistological examination showed a hard tissue-like appearance and inflammatory cell infiltration. The R_mCT^® images agreed with pathohistological finding on the days 50 after pulpotomy. Our research suggested that the R_mCT^® has made it possible to scan serial imaging of small animals such as experimental rats under anesthesia. Since its higher resolution enables clear observation of the entire hand of the rat, it is possible not only to compare the effectiveness of each pulpotomy method, but also to serially observe a single experimental animal during the process post-pulpotomy healing, growth, and aging. This study answered well for the animal protective law that to use the least of animal to got the best result.