Objective: The aim of this in-vivo study was to evaluate the changes in canal volume after root canal preparation with three different file systems i.e. OneShape, Standard Mani Silk and Blue Flex Acerfile using CBCT and 3D reconstruction. Method: Forty-five permanent mandibular premolar teeth were selected and divided into three experimental groups i.e. Group I (OneShape), Group II (Mani Silk File) and Group III (Blueflex Acerfile) compromising of fifteen teeth each. CBCT scans before and after root canal preparation were taken and stored in DICOM format. The DICOM files were sent to the second workstation for 3D reconstruction and pulp segmentation with BlueSkyPlan software. Segmented models were exported as STL file. Using 3D MeshMixer software, canal volume from CEJ to the apex of the tooth before and after preparation were calculated and results of the structure were obtained in mm³ in tabulated form. Student’s t test for paired data was used to determine statistically significant difference between the before and after canal volumes. One way ANOVA test was used to determine statistically significant differences in the percentage of canal volume increase between the groups and Tukey’s post-hoc test was used for paired comparison. Result: The comparison between initial canal volume of the canal prior to the instrumentation procedureand the final volume after preparation showed statistically highly significant difference in all three groups(p<0.001). Statistically significant difference was found betweenOneShape file,Standard Mani Silk file and Blueflex Acerfile (p<0.05). Conclusion:Blueflex Acerfileshowed higher increase in the root canal volume as compared to OneShape file, while Standard Mani Silk file showed minimum change in the canal volume during preparation