The aim of this study was to compare the post-operative pain after the use of manual or rotary instrumentation techniques of the same system (ProTaper Universal, Dentsply Maillefer, Tulsa Dental, TN, USA) in mandibular posterior teeth after one-visit root canal treatment after 6, 12, 24, 48, 72 hours and 7 days from the end of the endodontic treatment using numerical rating scale (NRS).Participants and Methods: Forty-six patients with vital or non-vital, mandibular posterior teeth without periapical pathosis, were endodontically treated in a single visit. The patients were randomly divided into two groups, 1 and 2, each consisted of 23 patients. Instrumentation was done using either manual ProTaper files for group 1 or rotary ProTaper files for group 2. Irrigation was done using 2.6% NaOCl and 17% EDTA, finally the canals were flushed with distilled water. Results: showed no statistically significant difference in the prevalence of postoperative pain after 6, 12, 24, 48, 72 hours and 7 days between the two groups. Conclusions: Post-obturation pain was similar on using manual or rotary ProTaper Universal systems in cases of vital and non-vital mandibular posterior teeth. The intensity of pain is at its highest levels during the first 24 hours and decreases gradually by time.