In order to meet the demands of high-throughput metabolomics analysis, we established urine sample preparation method on 96-well plates and an efficient liquid chromatography (LC) coupled to high-resolution mass spectrometry (MS) method using the latest model of the Vanquish™ Tandem LC system. The separation of urine metabolites was performed using two Hypersil GOLD™ C18 columns (150 x 2.1 mm, 1.9 µm) operating in parallel and running with a fast linear gradient of acetonitrile containing 0.1% formic acid, ramping from 5% to 95% in 10 minutes, alternating between the two columns. Dual-column operation with column reconditioning maintains full occupancy of the MS instrument (MS acquisition of column A during re-equilibration of column B). MS detection was realized on a Q Exactive™ HF mass spectrometer operating in positive and negative electrospray ionization acquisition modes. Data quality consistency was assessed through various pooled urine quality control samples injected on both columns processed by principal component analysis. To prevent possible drift in retention time across the columns, various reference index markers eluting across the gradient were analyzed at the beginning and the end of the 96-well plate series on each column. Retention index values were calculated to provide reproducible results over time. In addition, we have assessed the 13C yeast extract (TruQuant IQQ Workflow Kit, IROA®) to obtain a more robust method for metabolite identification and quantification.