An inter-laboratory comparison exercise was organized among European laboratories, under the aegis of EU COST Action 636: “Xenobiotics in Urban Water Cycle”. The objective was to evaluate the performance of testing laboratories determining “endocrine disrupting compounds” (EDC) in various aqueous matrices. As the main task three steroidal estrogens: 17α-ethinylestradiol, 17β-estradiol and estrone were determined in four spiked aqueous matrices: tap water, river water and wastewater treatment plant influent and effluent using GC-MS and LC-MS/MS. Results were then compared and discussed according to the analytical techniques applied, the accuracy and reproducibility of the analytical methods and the influence of the sample matrices. Out of a total of fourteen European laboratories that participated in the exercise, eleven laboratories submitted results. Statistical evaluation of results revealed that the number of outliers in this exercise was low taking into account the complexity of sample matrices and the differences in analytical methods applied. Consensus values are also in good agreement with the spiked concentrations for each compound, with the exception of the wastewater influent sample for which the obtained values differed significantly. This was in agreement with standard deviation of repeatability, inter-laboratory standard deviation and standard deviation of reproducibility. Overall the results obtained in this inter-laboratory exercise reveal a high level of competence among the participating laboratories for the detection of steroidal estrogens in water samples indicating that GC-MS as well as LC-MS/MS can equally be employed for the analysis of natural and synthetic hormones.