Among all the human biological fluids used for disease biomarker discovery or clinical chemistry, urine stands out. It can be collected easily and noninvasively, it is readily available in large volumes, it is typically free from protein contamination, and it is chemically complex-reflecting a wide range of physiological states and functions. However, the comprehensive metabolomic analysis of urine has been somewhat less studied compared to blood. Indeed, most published metabolomic assays are specifically optimized for serum or plasma. In an effort to improve this situation, we have developed a comprehensive, quantitative MS-based assay for urine analysis. The assay robustly detects and quantifies 142 urinary metabolites including 28 amino acids and derivatives, 17 organic acids, 22 biogenic amines and derivatives, 40 acylcarnitines, 34 lipids, and glucose/hexose, among which 67 metabolites are absolutely quantified and 75 metabolites are semiquantified. All the analysis methods in this assay are based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using both positive and negative-mode multiple reaction monitoring (MRM). The recovery rates of spiked urine samples at three different concentration levels, that is, low, medium and high, are in the range of 80% to 120% with satisfactory precision values of less than 20%. This targeted metabolomic assay has been successfully applied to the analysis of large numbers of human urine samples, with results closely matching those reported in the literature as well as those obtained from orthogonal analysis via NMR spectroscopy. Moreover, the assay was specifically developed in a 96-well plate format, which enables automated, high-throughput sample analysis. The assay has already been used to analyze more than 1800 urine samples in our laboratory since early 2019.