Mass spectrometry-based proteomics is fast growing and provides a powerful set of technologies, with the potential to revolutionize health care and enable precision medicine. The Evosep One is specifically designed for high throughput applications, with a focus on clinical analysis of large sample cohorts.For a widespread adoption of proteomics in the clinic, the entire workflow from sample preparation through LC-MS and data analysis needs to be fast and robust. To meet these requirements, we introduce an end-to-end workflow from protein lysate to peptides loaded on Evotips ready for injection on the Evosep One. Furthermore to facilitate high throughput analysis in the clinical laboratory, we have developed a new standardized method the Evosep One allowing 500 samples analyzed per day.