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A high-performance liquid chromatography/electrospray ionization/tandem mass spectrometry (LC/MS-MS) method was developed, validated and used to characterize pharmaceutical inputs in the Grand River watershed, Ontario, Canada. Twenty-seven antibiotics and neutral pharmaceuticals were extracted from aqueous environmental samples in a single step using 13C6-labeled sulfamethazine phenyl as a method surrogate and analyzed by LC/MS-MS. Method detection limits were in the range of 20-1,400 ng/L for the compounds of interest with recoveries from 51 to 130%. Using this method, pharmaceutical profiles in tributaries flowing into the Grand River were investigated using samples collected from seven agricultural sites and one urban site. Quality control data collected during the 5-month field study period showed good method performance. Concentrations of pharmaceuticals in source water with heavy agricultural input and surface waters downstream with urban inputs were determined to examine the effect of agricultural and urban inputs to surface water quality. Only human prescription drugs were found from urban site samples taken at both high-flow (spring and fall) and low-flow (summer) events in 2003, indicating minimal agricultural input. We also found for the first time the presence of the ionophore monensin in the environment as well as the occurrence of the human prescription drug carbamazepine in surface waters receiving primarily agricultural inputs. Possible causes and analytical solutions for some poor recoveries are discussed with respect to extraction pH, total organic carbon and metal ions in the water samples. The ability to extract 27 pharmaceuticals in a single solid-phase extraction step from diverse environmental matrices such as agricultural tile drain, tributary and surface water samples, followed by a single LC/MS-MS analysis, enabled the effective delivery of quality data.