Immunohistochemical assessment of 5-bromo-2-deoxyuridine (BrdU) in tissue sections is a widely used method to evaluate cell proliferation in vivo. However, this method requires timeconsuming preparation of paraffin sections and laborious counting of BrdU-labeled nuclei on multiple sections. Here, we report the development of a rapid and reliable method to quantitate BrdU incorporation in intestinal and liver tissues utilizing a dot-blot method. In vivo models of colon or liver proliferation were utilized to analyze the usefulness and reliability of this new method. Mice were sacrificed after BrdU injection, and genomic DNA was isolated from the tissues, denatured, and dot-blotted onto a nitrocellulose membrane. The incorporated BrdU was detected with a BrdU monoclonal antibody, and the signal intensity was densitometrically quantified. Results were compared with BrdU index determined by conventional immunohistochemistry on the same tissue samples. The patterns of colonic BrdU incorporation during fasting and refeeding, measured by the dot-blotting method and the immunohistochemical method, were similar. The BrdU incorporation in the regenerating liver after partial hepatectomy, evaluated by these two different methods, showed a strong correlation (R² = 0.91, p < 0.01). In addition, the inhibition of colon proliferation by the phosphoinositol 3-kinase (PI3K) inhibitor, wortmannin, was demonstrated by this dot-blotting method. In conclusion, the dot-blotting technique described in this report provides an accurate, more efficient, and possibly more reliable method for the assessment of in vivo proliferation compared with conventional immunohistochemical determination of BrdU labeling index.