The occurrence of endocrine disrupting chemicals (EDCs) in aquatic environments has long been a concern because of their threat to human and aquatic health. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) were optimized and compared for the detection of potential EDCs in order to examine the advantages and limitations of these commonly used instrumental methods in environmental analysis. The comparison between the two methods indicated that the performance and analytical results were not significantly different, except for the analysis of legacy organochlorine pesticides, such as dichlorodiphenyltrichloroethane (DDT) and its metabolites, where GC-MS/MS outperformed LC-MS/MS. However, because of the very low water solubility of these compounds they are less likely to be found in surface water samples, and they were therefore removed from the list of target analytes in the LC-MS/MS method. On the other hand, by using LC-MS/MS, highly water-soluble EDCs such as estrogens and their conjugates were simultaneously analyzed with the currently registered pesticides without the need for derivatization. In this study, an optimized procedure for solvent exchange of sample extracts originally dissolved in ethyl acetate (for GC-MS/MS) is described in order to prepare them for LC-MS/MS analysis. Quantitative analysis using LC-MS/MS was developed for the detection of potential EDCs including estrogens, pesticides, and industrial chemicals. Scheduled selected reaction monitoring acquisition in LC-MS/MS assured the optimum sensitivity of trace analysis. Electrospray positive and negative ionizations were performed in a single run by using wrong-way-round ionization employing a pH 10 mobile phase. Limits of detection ranging from 0.4 to 6 ng L⁻¹ for all the target compounds could be achieved using the developed LC-MS/MS method. Recoveries (n = 3) for most of the compounds were above 70% with a relative standard deviation below 20%. The validated LC-MS/MS method was applied to natural river water samples and the results showed no significant difference from those obtained using GC-MS/MS.