The purpose of this study was to evaluate the topography of new and used ProTaper rotary nickel-titanium (NiTi) instruments by using atomic force microscope. Four new and four used size S1, S2, F1, and F2 instruments were selected for this study. New and used instruments were analyzed on 11 points along a 3-mm section at the tip of the instrument. Quantitative measurements according to the topographic deviations (root mean square) were recorded. Data were analyzed by paired samples t test. Mean root mean square values for used ProTaper instruments were higher than the new ones, and the difference between them was statistically significant. The results of this study showed that used ProTaper instruments demonstrated more surface deformation and wear.