An analytical procedure for the determination of steroid estrogens and their conjugates was developed and applied to aqueous environmental samples. The analytes of 15 compounds were solid-phase extracted and fractionated into two fractions: one containing unconjugated (free) steroids and the other containing conjugates (sulfates and glucuronides). Identification and quantification were carried out using liquid chromatography coupled with tandem mass spectrometry. The recoveries for each compound ranged from 57 to 116% and reproducibilities represented as RSD ranged from 2.9 to 17%. Some of the sulfates and free compounds were detected in environmental samples, whereas most of the conjugates were below the detection limits.