In this work, a fully automated parallel LC column method was established in order to perform orthogonal hydrophilic interaction chromatography (HILIC) and reversed-phase (RPLC) chromatography within one analytical run for targeted quantitative mass spectrometric determination of metabolites from central carbon metabolism. In this way, the analytical throughput could be significantly improved compared to previously established dual separation work flows involving two separate analytical runs. Two sample aliquots were simultaneously injected onto a dual column setup columns using a ten-port valve, and parallel separations were carried out. Sub 2 μm particle size stationary phases were employed for both separation methods. HILIC and RPLC eluents were combined post column followed by ESI-MS/MS detection. The orthogonal separations were optimized, aiming at an overall separation with 2 retention time segments, while reversed-phase separation was accomplished within 5.5 min; metabolites on the HILIC phase were retained for a minimum time of 6 min. The overall run time was 15 min. The setup was applied to the quantification of 30 primary intercellular metabolites, including amino acids, organic acids, and nucleotides employing internal standardization by a fully (13)C-labeled yeast extract. The comparison with HILIC-MS/MS and RPLC-MS/MS in separate analytical runs revealed that an excellent analytical performance was achieved by the parallel LC column method. The experimental repeatability (N = 5) was on average <5% (only for 2 compounds >5%). Moreover, limits of detection for the new approach ranging from 0.002-15 μM were in a good agreement with ones obtained in separate HILIC-MS/MS and RPLC-MS/MS runs (ranging from 0.01-44 μM).