Background: To introduce a new method for measurement of surface roughness of the endodontic instrument, before and after instrumentation, using the Field Emission Scanning Electronic Microscope (FE-SEM) combined with the ImageJ software. Methods: Twenty J-shape resin blocks were divided into two groups, ten blocks of each group. Simulated root canal inside the resin block was 16 mm length, 60 0 angle of curvature, and radius of 4.5 mm. Ten WaveOne Gold Primary and 10 Reciproc Blue R25 instruments were used for root canal instrumentation. The instruments were scanned before and after instrumentation with special molds made to ensure the same areas at the point located 3 mm from the tips of the instruments using the FE-SEM. These scanned images were analyzed using the ImageJ. The arithmetical mean roughness (R a), root mean square roughness (R q), and the average distance between the highest peak and lowest valley in each sampling length (R z) were calculated by ImageJ for quantitative analyses. The paired-t test was performed to analyze the data using the SPSS 22.0 at the significance of .05. Results: Almost all surface roughness values were decreased. However, these decreases were not statistically significant (P > .05). Conclusions: The FE-SEM combined with the ImageJ was the reliable and appropriate modality for measurement surface roughness of instruments.