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The aim of this study was to optimize the solid-phase extraction method of several antibiotics in order to determine their concentration in wastewater (influent and effluent) and manure samples. The selected antibiotics for the present study were ampicillin, ceftazidime, cefepime, imipenem, piperacillin/tazobactam, tetracycline, erythromycin, ciprofloxacin, norfloxacin, vancomycin, gentamicin, sulfamethoxazole/trimethoprim. The optimum extraction conditions were established using a three- and four-factor Box-Behnken design under response surface methodology. The extraction of the antibiotics from liquid matrices was performed on several types of cartridges (Oasis HLB, Strata-X and HyperSep Retain PEP). With Oasis HLB cartridges were obtained the best recovery degrees (74.81 and 105.39% with the exception of tazobactam 58.70% and gentamicin 51.57%). This type of cartridges was further used for all of the samples collected for this study. In the case of the manure samples were tested two different extraction techniques: ultrasounds- and microwave-assisted solvent extraction, the first one being the most efficient. All the extractions obtained during the present study were evaluated by high-performance liquid chromatography with two detectors, diode array and mass spectrometer in positive ionization mode.