During the last five decades, the consumption of estrogens (natural and synthetic) 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. Estrogen 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 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 hormones (estrone [E1], 17aestradiol [17a-E2], 17bestradiol [17b-E2], 17aethynylestradiol [EE2] and estriol [E3]) using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). These 5 hormones were chosen because of their strong endocrine-disrupting potency in river and wastewater. The sample preparation consists of an extraction on Oasis HLB cartridges followed by purification on Florisil cartridges. The use of 4 perdeuterated hormones as internal standards spiked before the extraction step allows controlling matrix effects. The LC-MS-MS conditions include the use of 2 fragment ions for each compound, one for the quantification and one for the identity confirmation. The complete protocol and its performance are presented in detail. Limits of quantification for each compound are around 1 ng/L in river waters (for 100 mL sample volume, 250 µL of final extract and 10 µL injected). Recoveries higher than 60 % for E1 and EE2 and higher than 42 % for E3, 17a and b-E2 were obtained for quintuplet samples of surface water, influent and effluent from WWTP. Relative standard deviations (RSD) were found lower than 29 % except for E3 in influent samples with a RSD up to 80 % (result that we need to further verify). However, except for E3 in influent sample, the analytical protocol is satisfying and the use of perdeuterated hormones as internal standards appears to be an efficient method to correct for the lower recoveries.