In vitro bioassays have been successfully used to screen for estrogenic activity in wastewater and surface water, however, few have been applied to treated drinking water. Here, extracts of source and treated water samples were assayed for estrogenic activity using T47D-KBluc cells and analyzed by liquid chromatography-Fourier transform mass spectrometry (LC-FTMS) for natural and synthetic estrogens (including estrone, 17β-estradiol, estriol, and ethinyl estradiol). None of the estrogenswere detected above the LC-FTMS quantification limits in treated samples and only 5 source waters had quantifiable concentrations of estrone, whereas 3 treated samples and 16 source samples displayed in vitro estrogenicity. Estrone accounted for themajority of estrogenic activity in respective samples, however the remaining samples that displayed estrogenic activity had no quantitative detections of known estrogenic compounds by chemical analyses. Source water estrogenicity (max, 0.47 ng 17β-estradiol equivalents (E2Eq) L−1) was below levels that have been linked to adverse effects in fish and other aquatic organisms. Treated water estrogenicity (max, 0.078 ng E2Eq L−1) was considerably below levels that are expected to be biologically relevant to human consumers. Overall, the advantage of using in vitro techniques in addition to analytical chemical determinations was displayed by the sensitivity of the T47D-KBluc bioassay, coupled with the ability tomeasure cumulative effects of mixtures, specifically when unknown chemicals may be present.