Objectives To optimize a microtomographic (micro-CT) technique to quantitatively evaluate the effectiveness of contemporary caries-excavation techniques. Methods A beam-hardening curve was obtained from an initial reconstruction of a wedge-shaped hydroxyapatite (HAp) block and fitted with a 5th order polynomial function, after which each micro-CT tooth slice was corrected accordingly. Calibration of the 8-bit gray values into mineral-density values was obtained by scanning, reconstructing and processing volume of interests (VOIs) of HAp phantoms with different mineral densities (0.25, 0.75, 3.14 g/cm 3). One carious tooth was scanned before and after caries removal with an experimental enzyme-based gel. After reconstruction, a 3D-median filter was applied to each micro-CT slice, and a connected threshold grower algorithm was used to blank-out undesired structures in each slice. Volume rendering with a look-up-table (LUT), based on mineral densities, was accomplished for the tooth before and after caries removal. Finally, the actual volume of excavated tissue was quantified. Results Correction for beam hardening produced tooth slices with relatively homogeneous gray values along the whole area of enamel and dentin. Accurate mineral-density values were obtained for enamel, dentin and carious regions (2.89, 1.74 and 0.27 g/cm 3, respectively). After pre-processing (3D-median filtering and connected threshold grower algorithm), acceptable segmentation of carious dentin based on gray values was accomplished (Otsu method, gray value = 75 or mineral density = 1.12 g/cm 3), from which quantitative volumetric parameters were calculated. Significance Accurate calibration, standardization of scanning and reconstruction steps and adequate pre-processing of micro-CT slices allowed detailed volumetric calculation of caries-excavation techniques.