The effect of acid etching on root dentine was analyzed with Atomic Force Microscopy using a liquid cell that allowed image acquisition while the sample was kept in contact with the etching solution. The time evolution of the etching process was evaluated both qualitatively and quantitatively. Image sequences against etching time were obtained showing a clear trend of surface cleaning and opening of dentine tubules. Image analysis methods were employed to quantify the changes on the dentine surface, through the detection and morphological measurement of dentine tubules over time. The measurements indicated an overall increase in the diameter of the tubules against time, with a saturation value between 3.5 and 5.5 microm reached after approximately 300 seconds.