The US EPA Method 1694 (which is specific to the analysis of pharmaceuticals and personal care products) was evaluated for analysis of selected hormones: 17α- and 17β-estradiol, estriol, estrone, 17α-ethynylestradiol, diethylstilbestrol, equilin, equilenin, testosterone, progesterone and norgestrel in filtered sewage influent/effluent and seawater. The extraction of hormones from fortified water matrices with HLB Oasis solid phase extraction cartridges resulted in good recovery efficiencies: 86–113% for seawater and 80–114% for sewage samples with a relative standard deviation of 6–16% (seawater) and 7–14% (wastewater). Natural and synthetic estrogens (17α- and 17β-estradiol, estriol, estrone, 17α-ethynylestradiol, diethylstilbestrol, equilin, equilenin) were separated with a Gemini-NX C18 (Phenomenex, Inc.) column and detected by Liquid Chromatography Tandem Mass Spectrometry (LC-MS-MS) in negative ElectroSpray Ionization (ESI) multiple reaction monitoring (MRM) mode. Three other hormones (testosterone, progesterone and norgestrel) were separated using an Xterra MS C18 (Waters Corp.) column along with the Group I compounds of the EPA Method 1694 and detected by MS–MS in ESI positive mode. Most compounds were quantified by isotope dilution with matched labeled internal standards. The method was applied to the analysis of effluent and influent from wastewater treatment facilities in South Carolina, USA, and surface seawater samples from Charleston Harbor, South Carolina. Only estrone was detected in surface seawater at a concentration of 0.5 ng L−1, which was below the method reporting limit of 1 ng L−1. Estriol, estrone and testosterone were found in influent samples at 155–179 ng L−1, 27–28 ng L−1 and 41–49 ng L−1, respectively. The estrone concentration was 24 ng L−1 in an effluent sample from one of the treatment plants. This study demonstrates that the US EPA Method 1694 can be successfully used for the analysis of natural and synthetic hormones along with the pharmaceuticals and personal care products (PPCPs) currently listed in the method.