This paper presents an analytical method applied to the determination of 3 natural steroid hormones, estrone, 17 beta-estradiol, and 16 alpha-hydroxyestrone, and the contraceptive estrogen, 17 alpha-ethynylestradiol, at the sub-ng/L level in water samples [surface water and wastewater treatment plant (WWTP) samples]. The solid-phase extraction conditions were optimized using C18 cartridges. Identification and quantification were performed using a column liquid chromatographic/tandem mass spectrometric system with electrospray ionization in the negative mode. Before analyzing steroids in complex matrixes, statistical tools permitted a fine validation of the analytical method, and performance was evaluated for spring water in terms of recovery, specificity, trueness, repeatability, and intralaboratory reproducibility. The results showed the accuracy of the method, and limits of detection (LOD) ranged between 0.02 and 0.21 ng/L. The determination of steroids in WWTP effluents, which contain high levels of organic matter, required an additional purification step on silica cartridges. The high efficiency of the purification was proved with LOD < 0.3 ng/L from 200 mL sample. Specificity of the entire analytical procedure was shown by repeatable recoveries at low and high levels of the calibration range.