Method development and validation for the analysis of 20 hormones (including estrogens, androgens and progestagens compounds) in various aqueous matrices. During the last twenty years, consumption of hormones compounds (natural hormone or synthetic analogue) mainly for human medicine has considerably increased. Both androgens, progestogens and estrogens compounds are usually not entirely metabolized and reach aquatic environment mainly via effluents of wastewater treatment plants. All of them can be considered as endocrine disrupting compounds because of undesirable effects in biota. Hence, highly sensitive and selective analytical method is needed to identify and quantify these emerging contaminants in various environmental compartments. The aim of this communication is to show the different steps for development and validation of an analytical method for 20 hormonal compounds (estrone [E1], 17 estradiol [aE2], 17 estradiol [bE2], 17 ethynylestradiol [EE2], estriol [E3], androstenedione [ANDRO], androsterone [ANDROSTER], cortisone [CORT], cortisol [CORT-OH], dexamethasone [DEXA], dienestrol [DIEN], diethylstilbestrol [DES], drospirenone [DROSPI], epitestosterone [ETESTO], levonorgestrel [LEVO], medroxyprogesterone [MEDRO], megestrol acetate [MEGAC], norethindrone [NORET], progesterone [PROG], testosterone [TESTO]) in aquatic environments chosen because of their strong or potential endocrine-disrupting potency in river and wastewater. The sample pretreatment procedure that has been determined with a simple experimental screening design (Statgraphics software from SigmaPlus was used for data processing) consists of an extraction on polymeric based phase followed by purification on normal phase (Florisil). Eighteen stable isotope labelled compounds were used as surrogates to overcome matrix effects generated during whole analytical process. Ultra high performance liquid chromatography (UHPLC) coupled to electrospray (both positive and negative ionizations) tandem mass spectrometry is performed in the multiple reaction monitoring (MRM) mode, so molecule identity can be confirmed by two quantitation and confirmation transitions according to the EC Commission Decision (2002/657/CE). Accuracy and limits of quantification in intermediate fidelity conditions (intra-laboratory reproducibility) have been evaluated in accordance with the French standard (NF T90-210, 2009) on different aqueous matrices.