Abstract Aim: In-vitro comparative evaluation in the removal of Smear layer and Debris during root canal instrumentation using Two Rotary and Two Reciprocating file systems with Scanning Electron Microscope analysis. Materials & Method: Eighty extracted human permanent mandibular single rooted premolar teeth were collected and randomly divided into four groups, 20 teeth per group based on type of files used for root canal instrumentation. Group 1: ProTaper Gold (PTG) and Group 2: TruNatomy (TN) Rotary file systems. Group 3: Wave One Gold (WOG) and Group 4: Reciproc Blue (RB) Reciprocating file systems. All root canals were irrigated with Sodium hypochlorite and Ethylene-diamine-tetra acetic acid during canal instrumentation. The Coronal, Middle and Apical-third’s of root canals in all specimens were then examined using Scanning Electron Microscope (SEM) for the presence (or) absence of Smear layer and Debris. Results: One-Way Anova and Tuckey Post-hoc tests were used for statistical analysis. Statistically significant difference was seen in the removal of debris and smear layer among the four groups, P≤ 0.05. Group 3 (Wave One Gold) followed by Group 2 (TruNatomy) showed the presence of least or minimal smear layer and debris at all 3-levels of root canals. Whereas, Group 1 (ProTaper Gold) showed the presence of maximum smear layer and debris at all 3-levels of root canals. Conclusion: Wave One Gold Reciprocating file showed maximum efficiency and ProTaper Gold showed least efficiency in the removal of both Smear layer and Debris at all 3-levels of root canals compared to other file systems used.