During the last five decades, the consumption of estrogens for human medicine (contraception, management of menopausal and post-menopausal syndrome, physiological replacement therapy in deficiency states and treatment of prostate and cancers) and animal farming (growth promoters, developers of single-sex populations of fish in aquaculture) has considerably increased. Estrogens are usually not entirely metabolized and they reach aquatic environments mainly via the effluents from wastewater treatment plants (WWTP). The lack of knowledge on the toxicity and level of exposition of these compounds and their impact on ecosystems and human health has raised public concern about their occurrence in the environment. Thus, efficient analytical protocols are needed to identify and quantify these emerging contaminants in various environmental compartments. In this study, we developed an analytical protocol for the determination of five estrogenic hormones (estrone [E1], 17aestradiol [17a-E2], 17bestradiol [17b-E2], 17aethynylestradiol [EE2] and estriol [E3]) including their conjugated forms in various aqueous matrices (rivers, WWTP influents, WWTP effluents) using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). These 5 hormones were chosen because of their strong endocrine-disrupting potency in surface waters. The analytical protocol was validated according to the French standard NF XPT 90-210 (AFNOR, 1999). The procedure included the verification of linearity, the determination of limits of detection (LD) and quantification (LQ) and the evaluation of the specificity of the method (absence of matrix effects for various sample types). We also studied the recoveries and repeatability for the extraction and purification steps and for the hydrolysis of conjugated forms, and we evaluated preservation of wastewaters during 6 days.