We compared methods of determination of 17α-ethinyloestradiol (EE2) in surface water in Kielce, Poland. EE2 plays a role as a contraception agent and it is commonly used as an active agent in contraception pills. During studies three methods of EE2 determination were checked: TLC, HPLC, and ELISA assay. Samples of contaminated water were taken from eight points on four rivers crossing Kielce city, tap water, and sludge in a sewage treatment plant (STP). Assays revealed that EE2 is present in river water in amounts of 3.2 ng/L to 6.29 ng/L. The EE2 removal process in STP is ineffective, removings only 1.3%. EE2 was not detected in Kielce’s tap water. No correlation between fecal microbial contamination and EDS presence was determined.