Objective: This study developed a computer image analysis technique as a quantitative means to measure changes in dental stain after brushing with various dentifrices. Method and materials: Enamel specimens with naturally occurring mature stain were cut from bovine incisors. The specimens were subjected to in vitro toothbrushing with one of the four tested groups, consisting of two dentifrices that make claims of stain removal (Aquafresh Whitening and Rembrandt Sensitive), a regular dentifrice, (Aquafresh Triple Protection), and water. Digital images of stain specimens were recorded uner standardized lighting conditions and analyzed with an image analysis software. The area-intensity stain determinant, which accounted for the reflected intensity and the corresponding areas of stain, was computed. Stain removal efficacy was calculated based on the difference in area-intensity stain determinant before and after brushing. Results: Brushing with any of the tested dentifrices removed more stain than did brushing with water along. The fining that brushing with a regular dentifrice resulted in higher stain removal efficacy than brushing with water seems to indicate a role for abrasivity. Aquafresh Whitening had a higher stain removal efficacy than did Rembrandt for the removal of mature calcified stain used in this study. However, there were certain stains that none of the dentifrices removed. Conclusion: Computer image analysis provides an objective and quantitative measurement to distinguish in vitro stain rem oval efficacy of dentifrices.