The present study describes the development, validation and a practical application of a fully automated analytical method based on on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) for the simultaneous determination of 19 sulfonamides, including one acetylated metabolite, in different water matrices. MS/MS detection was carried out in a quadrupole-linear ion trap (QqLIT) mass analyzer. Target compounds were identified in the selected reaction monitoring (SRM) mode, recording two transitions between precursor ions and the two most abundant product ions. The method developed was applied to evaluate the occurrence of the target antibiotics in different water samples: influent and effluent water from waste water treatment plants (WWTP), ground water and surface water. Under optimal conditions, the method detection limits achieved were in the range 0.05-7.84 ng/L for WWTP influent water, 0.01-6.90 ng/L for WWTP effluent water, 0.02-5.13 ng/L for ground water and 0.02-4.52 ng/L for surface water samples. The instrumental repeatability, expressed as RSD, was usually below 10% for the different water matrices. Results showed the wide presence of sulfonamides in the four types of water, including one acetylated metabolite, with maximum concentrations up to 855 ng/L corresponding to sulfapyridine in an influent waste water sample near a densely populated urban area.