Objective: To compare the effect of glide path established by size 20/0.02 taper hand K-file and size 20/0.04 taper rotary file, on the amount of debris extruded apically during root canal instrumentation by rotary ProTaper Universal (S1-F2) and reciprocating WaveOne (Primary) file systems. Materials and methods: Eighty mandibular molars with curved mesial roots and two separate canals were selected and randomly divided into two groups (n = 40) for glide path preparation using either size 20/0.02 taper hand K-files and size 20/0.04 taper rotary files. These groups were further divided into two (n = 20) for root canal instrumentation by ProTaper Universal and WaveOne files; Group 1: 20/0.02 + ProTaper, Group 2: 20/0.02 + WaveOne, Group 3: 20/0.04 + ProTaper, and Group 4: 20/0.04 + WaveOne. The debris extruded during instrumentation was collected in pre-weighed Eppendorf tubes and stored in an incubator at 70°C for 5 days. Tubes containing the dry, extruded debris were then weighed. One-way analysis of variance was applied to the weights obtained followed by Tukey's post hoc test for multiple comparison. Results: All the groups resulted in debris extrusion. Establishing a glide path with size 20/0.04 taper rotary files, resulted in less debris extrusion, compared to a glide path prepared by size 20/0.02 taper hand K-files (P 0.001). Conclusion: Creating a glide path with size 20/0.04 rotary file, reduced the debris extrusion in curved root canals during instrumentation by ProTaper Universal and WaveOne. Keywords: apical extrusion, glide path, instrumentation, ProTaper Universal, WaveOne