Abstract A parallel three-column LC–MS/MS system for quantitative high-throughput in vitro screens is described. The robust novel system is composed of three LC pumps, an autosampler and a triple quadrupole mass spectrometer used in combination with three valves and three analytical LC columns in parallel configuration. Two of the three valves work in unison to select which column receives the injection, and the third valve selects which column is to be in line with the mass spectrometer. Improved sample throughput is achieved without sacrificing chromatographic separation quality or sensitivity. To demonstrate the applicability of the system, pools of five compounds (phenacetin, S-mephenytoin, bufuralol, midazolam and clomethiazole) were analyzed, together with an internal standard. The results show that the sample throughput can be increased significantly by reducing analysis time to 3 min per sample as compared to 8 min with a general gradient single-column system. Analysis of the five compounds shows an accuracy of 81–108% and a precision (given as relative standard deviation) of 1.5–14%. The system was further applied to samples from a metabolic stability assay in liver microsomes.