Aim: The aim of the present study was to evaluate the efficacy of different instrumentation for removal of gutta-percha and sealers in endodontic retreatment. Materials and methods: A total of single-rooted mandibular premolars were collected. Access opening was performed in all specimens. The working length was determined by reducing 1 mm from the length of instruments that were seen reaching the apex. All the canals were dried with paper points. Lateral condensation technique was used to obturate the canals with gutta-percha and zinc oxide eugenol was used as a sealer. The specimens were randomly segregated into three experimental groups. Group I: ultrasonic retreatment tip, group II: R-Endo retreatment files, group III: Mtwo retreatment files. Roots were grooved into two halves with a diamond disc in a longitudinal, buccolingual direction. A stereomicroscope with ×40 magnification was used to image the quantity of gutta-percha and sealer on canal walls. Results: With ultrasonic retreatment tip, least amount (1.96 ± 0.32) of gutta-percha and sealer remained, followed by Mtwo retreatment files (2.84 ± 0.24) and R-Endo retreatment files (3.18 ± 0.63). A statistically significant inter-group difference among different instrumentation groups was demonstrated by ANCOVA analysis. Conclusion: In the present study, ultrasonic retreatment tip file systems was found to be more effective in the removal of root canal filling material, followed subsequently by Mtwo retreatment file system and R-Endo retreatment file system. Clinical significance: Thorough removal of filling material from the root canals is essential during root canal retreatment for probable cleaning and shaping of canal morphology. Thus, clinicians should be aware of the suitable and better instrumentation system that provides a clean and sterile root canal system without any debris.