Vanillylmandelic acid (VMA), as one of the most important catecholamine metabolites, is commonly used to aid in diagnosis of pheochromocytoma. This study develops a rapid and simple high-throughput LC-MS/MS method for the measurement of urinary VMA. Without sample pretreatment, the urine specimens were mixed with internal standard (IS) solution for direct analysis by LC-MS/MS in two minutes. VMA and VMA-d3 were detected in the multiple-reaction monitoring mode using the specific transitions m/z 197.0 -> 137.0 and 200.0 -> 140.0, respectively. This method was validated for consistent linearity from 1.0 to 250.0 mu M with CVs <= 3.12%, excellent recovery, good stability and low carryover. The lowest limit of quantification (LLOQ) was 0.125 mu mol/L for VMA with CV of 14.1%, and the lower limit of detection (LOD) was 0.025 mu mol/L. Intra-assay and inter-assay imprecision values (CVs) for VMA were all below 2.11%. Dilution linearity was investigated with a satisfied mean accordance of 105%. Method comparison of LC-MS/MS and microcolumn chromatography in our lab was performed and the reference interval was established in agreement with that of the Mayo Clinic. All these results demonstrate that this validated LC-MS/MS approach shows improved accuracy and reproducibility compared with microcolumn chromatography. The low sample volume, simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories. (C) 2015 Elsevier B.V. All rights reserved.