Objective: To evaluate the accuracy of the new endodontic planning software (3D Endo Dentsply Sirona) based on cone beam computed tomography (CBCT) to predetermine root canal lengths compared with measurements performed with an electronic apex locator (Raypex 6; VDW) ex vivo. Materials and methods: CBCT scans of forty extracted human maxillary (n = 20) and mandibular (n = 20) molars were taken, and root canal lengths were predetermined with the 3D Endo software using the apical foramen (AF) and the adjoining cusp as references. Root canal lengths were determined with the Raypex 6 using the same references. To evaluate the accuracy, absolute differences between both methods and the actual root canal length (gold standard) were calculated and statistically analyzed. Results: Differences between lengths measured with the 3D Endo and the Raypex 6 compared with the gold standard showed no significant differences (P = 0.879). Mean differences were 0.37 mm versus 0.35 mm in the maxillary molars, and 0.30 mm versus 0.31 mm in the mandibular molars. A total of 75.8% (3D Endo) and 79.1% (Raypex 6) of all measurements were within the limits of ± 0.5 mm. Both methods showed a tendency to result in short measurements (P 0.001). Conclusions: Within the limitations of this study, the 3D Endo software enables an accurate three-dimensional (3D) predetermination of root canal lengths. Keywords: 3D Endo, ERCLMD, CBCT, Raypex 6, root canal length determination