This project evaluated solid-phase extraction (SPE) combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine the trace amount of rosiglitazone in human urine. The analytical performance of four modes of LC–MS and tandem MS operation (atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), positive and negative ionization) was compared for two mass spectrometers, a triple-quadrupole and a quadrupole ion trap instrument. Rosiglitazone was extracted from urine using a SPE cartridge of 50mg C8 sorbent and acetonitrile used as the eluting solvent. Samples were then separated on a RP18 column interfaced with a tandem mass spectrometer. The recovery of rosiglitazone was greater than 91.2%. The urine assay combining SPE and LC–APCI-MS/MS of triple-quadrupole was proved a very selective and sensitive method for determination of trace rosiglitazone. The assay was linear over a wide range, with a lower limit of quantification of 0.1ng/mL using 1mL of urine. The intra- and inter-day precisions were <9.8% and <7.9%, respectively, and the accuracies were in the range 91.0–103.6%. The rosiglitazone concentration profile in human urine was also determined. The results of this study reveal the adequacy of SPE–LC–APCI-MS/MS method for analyzing rosiglitazone from diabetic patients’ urines. The concentrations of rosiglitazone were detected to range from 760 to 164pg/mL.