A variety of devices and methods have been described for the ex vivo evaluation of the several steps of the root canal treatment. These methodologies have been successfully used for many years; however, some inherent limitations such as the destruction of the specimen, encouraging the search for new methods able to produce improved results. The development of X-ray micro-computed tomography (µCT) has gained increasing significance in the study of dental tissues. µCT offers a noninvasive reproducible technique for three-dimensional assessment of the root canal system and it can be applied before and after all root canal treatment steps. The purpose of this study was to perform historical and critical analyses of the studies on root canal anatomy as well as the use of µCT as a method for evaluating the several steps of the root canal treatment.