Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is the golden standard for immunosuppressants analyses, where optimising throughput by parallel chromatography can reduce costs and turnaround time. We aimed to double our system throughput using a dual LC-MS/MS setup. Therefore, two independent UPLC systems were hyphenated to one triple quadrupole MS, with staggered injections from one autosampler on alternating columns. The method simultaneously measured the analytes tacrolimus, sirolimus, everolimus, and cyclosporin A in whole blood using isotope dilution, with a run time of 1.5 min. Using the dual LC-MS/MS system, net run-to-run time improved from 2.3 to 0.98 min per injection, where throughput increased from 26 to 61 injections per hour. For Performance Qualification, 1101 clinical samples were measured on the dual LC-MS/MS system in addition to the standard system, during a period of one month, and the results were compared using Passing Bablok regression and Bland Altman analysis. There was excellent agreement for all four analytes, with regression slopes of 0.98–1.02x and intercepts of -0.11–0.88 µg/L. Minor bias was demonstrated between the systems with mean differences from -0.93 to 1.43%. In conclusion, the throughput was doubled and idle MS time was reduced with good agreement to the standard system. Currently, the method is applied for clinical routine with frequent peak intensities of >180 injections per day.