An effective and practical method for the determination of macrolide antibiotics azithromycin, clarithromycin, erythromycin and roxithromycin in wastewater samples has been developed. The analytical method combines solid phase extraction followed by a chromatographic separation by hydrophilic interaction liquid chromatography (HILIC) coupled with an ion trap mass spectrometer utilizing the electrospray ionization technique. Detection of positively charged ions was performed in full scan mode from 500 to 900 m/z. The method detection limits and method quantification limits obtained were in the range of 2.03-7.59 ng L-1 and 6.08-23.84 ng L-1, respectively. Recoveries of solid phase extraction were obtained using SupelTM-Select HLB cartridges ranging from 85.76 % to 92.54 %. All target antibiotics were detected in 100 % of the collected raw influent samples with concentrations varying from 15 ng L-1 to 1849 ng L-1. Azithromycin, clarithromycin and erythromycin were also detected in 100 % of the treated water samples and roxithromycin was present in 96 % of the samples. The highest determined concentration in the treated water samples was 1404 ng L-1 of azithromycin. Based on the determined macrolide concentrations, removal efficiencies of individual wastewater treatment plants were calculated to range from 13 % to 100 %.