The mammary glands (MG) undergo rapid expansion of the ductal network during puberty in response to endocrine cues including the potent mitogenic effects of estrogen. The proliferation of mammary epithelial cells occurs in a spatially distinctive manner, where terminal end buds located at the ductal termini are the primary site of cell division. Here, we present a relatively high throughput approach to spatially assess epithelial cell proliferation in whole mouse MG using histochemical detection of 5-ethynyl-2′-deoxyuridine in conjunction with a standard curve-based data deconvolution technique to semiquantitatively measure proliferation via wide-field epifluorescent microscopy. This approach was validated against the “gold standard” of counting labeled nuclei from confocal images utilizing computer-assisted image analysis. Our method proved sensitive enough to describe the significant and spatially variable proliferative response to low-dose estrogen after 108 hours. This flexible method presents a timely and economical approach to obtaining spatial information regarding epithelial cell proliferation in the mouse MG.