Endodontic success hinges on a multifactorial interplay, with meticulous canal shaping, proper disinfection, and three-dimensional obturation being paramount. Among these factors, the amount of dentin remaining after instrumentation directly influences the biomechanical resilience and longevity of the tooth. This study aims to evaluate the impact of various rotary instrumentation systems, including single-file, two-file, and multiple-file configurations, on the remaining dentin thickness (RDT) following canal preparation. This was an in vitro study, original research article. Sixty mandibular premolar human extracted teeth were decoronated at the cementoenamel junction with a diamond disc. Samples were randomly assigned to three groups using a simple random sampling technique (n = 60). Group I - Single-file system (One Curve, MicroMega) (n = 20), Group II - Two-file systems (2Shape, MicroMega) (n = 20), and Group III - Multiple-file system (Hero Gold, MicroMega) (n = 20). Preoperative cone-beam computed tomography (CBCT) scans were obtained after the sample was mounted on a modeling wax sheet. The biomechanical preparation of canals followed the manufacturer's protocols for every system. Postoperative CBCT scans were obtained. Pre- and postoperative scans were compared at standardized depths (4 mm, 7 mm, and 11 mm) within the canals (coronal, middle, and apical thirds), allowing for a comprehensive assessment of RDT throughout the canal. According to one-way ANOVA, the highest mean was seen in Group I, followed by Group II and Group III. Hence, a statistically significant difference was found between all the groups. Post hoc Tukey's test was done for intergroup comparison. A single-file system preserved more dentin with less aggressive cutting compared to two-file and multiple-file systems.