High turbulence liquid chromatography (HTLC, or turbulent flow online extraction) and tandem mass spectrometry (MS/MS) methods for the determination of sitagliptin in human urine and hemodialysate were developed and validated to support clinical studies. A narrow bore large particle size reversed-phase column (Cyclone, 50 mm x 1.0 mm, 60 microm) and a BDS Hypersil C18 column (30 mm x 2.1 mm, 3 microm) were used as extraction and analytical columns, respectively. For the urine assay, the LLOQ was 0.1 microg/ml, the linear calibration range was 0.1 to 50 microg/ml, the interday precision (R.S.D.%, n=5) was 2.3-6.5%, and the accuracy was 96.9-106% of the nominal value. For the urine quality control samples (QCs), the intraday precision (R.S.D.%, n=5) and accuracy were 1.8-2.6% and 96.2-106% of the nominal value, respectively. The interday precision (R.S.D.%) for 56 sets of urine QCs over a 6-month period varied from 3.8% to 5.5% and the accuracy from 102% to 105% of the nominal value. For the hemodialysate assay, the LLOQ was 0.01 ng/ml, the linear dynamic range was 0.01-5.0 ng/ml, the interday precision was 1.6-4.1%, and the accuracy was 89.8-104% of the nominal value. For hemodialysate QCs, the intraday precision and accuracy varied from 2.3% to 8.9% and from 99.8% to 111% of the nominal value, respectively. These results demonstrated that both methods are selective, accurate, precise, reproducible, and suitable for quantifying sitagliptin in hemodialysate and human urine samples.