Bruker’s timsTOF Pro mass spectrometer for high sensitivity proteomics can now be integrated with the Evosep ONE separation device for high-throughput clinical proteomics. The Evosep One is a novel chromatography system for large cohort proteomics, optimised for >200 samples/day and >90% MS utilisation. Its LC peak widths of ~2 s for ~5-min gradients have been combined with the PASEF method on the timsTOF Pro with MS/MS rates >150 Hz. This novel combination delivers sensitivity (50 ng HeLa) and throughput for LFQ of ~1200 proteins in ~5 min. For large sample cohorts, this enables biomarker research and validation on >200 samples per day.