Background Until now, the insertion depth of the enteroscope during double-balloon enteroscopy (DBE) could only be estimated. However, the currently available methods have limitations, and development of newer, simple, and accurate modalities is needed. Objective To evaluate the accuracy of a novel method for evaluation of enteroscope insertion depth during DBE. Design Prospective, single-center cohort study. Setting Tertiary referral university hospital. Patients Fifty-one patients who had lesions found during 41 antegrade and 10 retrograde DBEs and treated by surgery were enrolled in this study. Interventions The length of the ligament of Treitz/ileocecal valve lesion was estimated by adding the forward enteroscope length during each cycle of passage and by calculating the overtube insertion length (every 5 cm of overtube advancement means 40 cm of enteroscope advancement based on preliminary observations) during DBE, respectively, and was evaluated at surgery. Main Outcome Measurements The length from the ligament of Treitz/ileocecal valve to the lesion. Results Surgical evaluation was used as the standard. Regardless of insertion route, the mean difference from surgery in evaluation of enteroscope insertion length between using the enteroscope method and the overtube method was 19 cm (range 0-50 cm) and 17 cm (range 0-60 cm), respectively (P > .05). Limitations Small number of patients with a case series study design. Conclusions Calculating the length of the overtube passage is accurate, and it is simple to estimate the insertion depth of the enteroscope during DBE, which is useful in clinical practice. © 2010 American Society for Gastrointestinal Endoscopy.