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In this study, the analytical method optimization for analysis of (beta-blockers including metoprolol (MET), propranolol (PRO), atenolol (ATE), sotalol (STL), timolol (TML) in wastewater were carried out. The effect of various parameters such as sample volume, pH, concentration, matrix and pre-treatment procedure was studied in order to find out the optimum conditions for their determination in SPE system. The presence of the compounds in Konya urban wastewater treatment plant influent and effluent samples was investigated and the eco-toxicological risk assessment was carried out. The extraction of the samples was performed with SPE system using Oasis HLB cartridges. The concentrations of the target compounds were determined using a liquid chromatograph equipped with MS-MS detector. STL, ATE, MET, PRO, TML were determined in the range of 43.5-468, 278-465, 28.3-92,