In this paper, development and optimization of new LC-MS method for determination of twenty selected hormones, human/animal and plant sterols in river sediments were described. Sediment samples were prepared using ultrasonic extraction and clean up with silica gel/anhydrous sodium sulphate cartridge. Extracts were analyzed by liquid chromatography-linear ion trap-tandem mass spectrometry, with atmospheric pressure chemical ionization. The optimized extraction parameters were extraction solvent (methanol), weight of the sediment (2 g) and time of ultrasonic extraction (3× 10 min). Successful chromatographic separation of hormones (estriol and estrone, 17α- and 17β-estradiol) and four human/animal sterols (epicoprostanol, coprostanol, α-cholestanol and β-cholestanol) that have identical fragmentation reactions was achieved. The developed and optimized method provided high recoveries (73-118%), low limits of detection (0.8-18 ng g(-1)) and quantification (2.5-60 ng g(-1)) with the RSDs generally lower than 20%. Applicability of the developed method was confirmed by analysis of six river sediment samples. A widespread occurrence of human/animal and plant sterols was found. The only detected hormone was mestranol in just one sediment sample.