A robust novel technology of parallel chromatography combined with tandem mass spectrometry was successfully applied to a biological matrix extract for analyte detection. The presented study shows how only by using an additional isocratic pump, a second column and a 10-port valve the throughput is twice of that of a conventional single column system with the same sensitivity. Analytes and matrix were separated and eluting peaks of the first column were detected while the second column was equilibrated. The system was tested and used for the determination of several drugs, metabolites and endogenous compounds (i.e., propiverine, talinolol, scopolamine and leukotrienes).