Updated publication reference for PubMed record(s): 31308252. We describe a method for global enrichment of active peptide hormones from blood plasma for both new hormone discovery and quantitative analysis. To extend the dynamic range of plasma proteomics, we employed high-resolution size exclusion chromatography to purify the peptide hormones and a powerful precipitation method to dissociate and remove bulk proteins prior to separation. Combining this method with high-sensitivity proteomic analysis, we characterized hormone abundance changes after intermittent fasting in humans.