OBJECTIVES: Rats are used as animal models for basic and applied research related to orthodontic tooth movement (OTM). The magnitude of mechanical force in OTM rat models mainly depends on the supporting capability of the periodontal ligament (PDL), which is highly associated with the root surface area (RSA). But the size of rat RSA remains unknown, which is the reason why there are still debates on the magnitude of mechanical force in OTM rat models. This study aimed to explore a method for measuring the RSA in rat molars. DESIGN: The maxillary and mandibular samples of rats were scanned by Micro-CT to generate three-dimensional (3D) images, followed by 3D reconstruction of every molar through Mimics Medical 21.0. Geomagic Wrap 2021 and Unigraphics NX 12.0 were utilized to smooth teeth surface and mark the cementoenamel junction (CEJ). Finally, the RSA in rat molars was measured. RESULTS: The results showed that for the six-, eight-, or ten-week-old rats, the average RSA of maxillary first, second, and third molars was 25.90 ± 2.29 mm CONCLUSIONS: Through 3D modelling, we provided data of rat RSA, and observed the trend of increasing RSA mean values with age. These data are pivotal for determining the magnitude of mechanical force required to move rat molars, especially when conducting research related to OTM using rat models.