Background: The primary objective of root canal preparation is to completely debride the infected content and provide a sterile space for obturation. The present study has compared hand and rotary instrumentation in reducing post-operative pain in root canal treated teeth. Materials & Methods: Sixty non vital mandibular molar teeth were divided into 2 groups. Group I teeth were instrumented with manual technique and group II teeth were instrumented with rotary technique using rotary ProTaper files. In both groups pain using visual analogue scale (VAS) and time taken for procedure was recorded after 12 hours, 24 hours, 48 hours and 72 hours respectively. Results: In group I, the pre-operative mean visual analogue scale pain (VAS) score was 6.8 and in group II was 6.9 after 12 hours, in group I was 6.4 and in group II was 5.3 after 24 hours, 5.3 in group I and 4.4 in group II after 48 hours, and 2.7 in group I and 1.3 in group II after 72 hours. The mean time taken for instrumentation in group I was 53.1 and in group II was 20.4. The mean time for obturation in group I was 42.7 and in group II was 21.6. The difference was significant (P< 0.05). Conclusion: Hand instrumentation resulted in more post-operative pain as compared to rotary technique. Key words: Hand instrumentation, Pain, ProTaper files.