Aims and Objectives:The success of root canal treatment depends on proper debridement, instrumentation, proper accessibility, and proper restoration. The presence of a smear layer is considered to be a significant factor. This in vitro study was conducted to assess the effect of the presence/absence of a smear layer on the microleakage of root canal filled teeth using different instruments and obturation methods.Materials and Methods:One hundred extracted mandibular premolars with closed apices and single roots were chosen and then divided into six groups, A to F, consisting of 15 teeth each. The control group included 10 teeth; 5 positive and 5 negative. The teeth were decoronated at the cementoenamel junction. Groups A, B, C, and D were instrumented with engine-driven rotary Protaper NiTi files. Groups E and F were instrumented with conventional stainless steel hand files. Groups A, C, and E were flushed with 3 ml of 17% EDTA to remove the smear layer prior to obturation. All teeth were flushed with 5.25% sodium hypochlorite solution and obturated with AH-Plus sealer with lateral condensation technique for Groups C, D, E, F and with thermoplasticized gutta-percha technique for Groups A and B. Using an electrochemical technique, leakages in the obturated canals were assessed for 45 days. The results were tabulated using Student's t-test (paired and unpaired t-test) with the Statistical Package for the Social Sciences Software Version 21 (IBM Company, New York, USA).Results:Group A showed the lowest mean value at intervals of 10, 20, 30, and 45 days. There was no current flow in the negative controls during the test period. There was leakage in the positive controls within a few minutes of immersion.Conclusion:The results showed that rotary instrumentation contributed toward an exceptional preparation of root canals compared to hand instrumentation. Elimination of the smear layer enhanced the resistance to microleakage; thermoplasticized gutta-percha obturation technique produced a better seal compared to the lateral condensation technique.