Originally adapted from the neurosphere assay, the nonadherent mammosphere assay has been utilized to assess early progenitor/stem cell frequency in a given population of mammary epithelial cells. This method has also been used to measure the frequency of tumorsphere initiating cells in both primary mammary tumors as well as in tumor cell lines. Although, the mammosphere assay has been used extensively in the mammary gland field, a standard method of quantifying and analyzing sphere growth in this assay has remained undefined. Here, we discuss the use and benefit of using a limiting dilution analysis to quantify sphere-forming frequency in primary mammary epithelial cells grown in nonadherent conditions.