Using effect directed analysis, the presence of estrogenic components in untreated and biologically treated oil sands process water (OSPW) was detected with the yeast estrogenic screening assay after fractionation with solid phase extraction followed by reversed phase high performance liquid chromatography. Comparison of the composition, as determined by electrospray ionization combined with high-resolution linear trap quadropole (LTQ)-Orbitrap Velos Pro hybrid mass spectrometry (negative ion) of selected estrogenic and nonestrogenic fractions identified compounds that were uniquely present in the estrogenic samples, biologically treated and untreated. Of the 30 most abundant compounds, there were 14 possible nonaromatic structures and 16 possible aromatic structures. Based on the published literature, the latter are the most likely to cause estrogenicity and were O2, O3 and O4 C17 to C20 compounds with double bond equivalents between 6 and 10 and chemical formulas similar to estrone- and estradiol-like compounds. This study shows exact formulas and masses of possible estrogenic compounds in OSPW. These findings will help to focus study on the most environmentally significant components in OSPW.