This study examined the effects of ionic strengths of NaCl (0.1, 0.3, and 1.0 M), pH (3, 7, and 11), and organic solvents (dichloromethane, diethyl ether, and methanol) on the extraction of estradiol at concentrations of 5.0 pg/mL in human serum. Methanol extracted almost 100% of the estradiol at a 5.0 pg/mL concentration, while ether and dichloromethane extracted only 73% or 70%, respectively, of the estradiol. The methanol extracted material was subjected to reverse phase high-performance liquid chromatography (HPLC) using 60% methanol and was found to elute at the same position as estradiol standard. These results suggest that methanol extraction of estradiol may prove useful in situations where estradiol occurs at concentration levels of ≥5.0 pg/mL, concentrations of great clinical significance in the detection and treatment of breast cancer.