Most analytical methodologies currently available for the determination of steroids in water only identify a few representative compounds (mainly estrogens). In this context, a multi-residue methodology based on liquid chromatography-tandem mass spectrometry was developed for the determination of 26 steroids including natural and synthetic estrogens, progestagens and androgens. The method described involves limited sample preparation as it includes a filtration followed by a single solid-phase extraction step using a C18 cartridge. The analytical procedure allows the determination of the target analytes in the lower ng/L range, with recoveries above 80%. The methodology was successfully applied to the analysis of steroids in several surface and ground-waters. In all the waters, estrogens, androgens and/or progestagens were determined.