Background/Aims: Excretion of urinary compounds in spot urine is often estimated relative to creatinine. For the growing number of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays of urine-excreted molecules, a fast and accurate method for determination of creatinine is needed. Methods: A high-throughput flow injection tandem mass spectrometry method for exact quantitation of creatinine in urine has been developed and validated. Sample preparation used only two-step dilution for protein precipitation and matrix dilution. Flow injection analysis without chromatographic separation allowed for total run times of 1 min per sample. Creatinine concentrations were quantitated using stable isotope dilution tandem mass spectrometry. Selectivity and coelution-free quantitation were assured by qualifier ion monitoring. Results: Method validation revealed excellent injection repeatability of 1.0% coefficient of variation (CV), intraday precision of 1.2% CV and interday precision of 2.4% CV. Accuracy determined from standard addition experiments was 106.1 +/- 3.8%. The linear calibration range was adapted to physiological creatinine concentrations. Comparison of quantitation results with a routinely used method (Jaffe colorimetric assay) proved high agreement (R-2 = 0.9102). Conclusions: The new method is a valuable addition to the toolbox of LC-MS/MS laboratories where excretion of urinary compounds is studied. The `dilute and shoot' approach to isotope dilution tandem mass spectrometry makes the new method highly accurate as well as cost-and time-efficient. Copyright (C) 2012 S. Karger AG, Basel