Proteomics is one of the "omics" disciplines that has provided molecular insights into the pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Here we describe a complete SWATH-MS workflow for the quantitative profiling of proteins extracted from peripheral mononuclear blood cells to investigate proteomic alterations in ME/CFS. This workflow covers all steps of sample preparation, data acquisition, and data analysis. We describe the process of generating a comprehensive spectral library from a pre-fractionated peptide reference sample followed by the acquisition of DIA data sets of individual samples using a 5600+ TripleTOF mass spectrometer. Examples of both library-based and library-free data analysis pipelines are presented based on the PeakView/MarkerView software package (commercial) and DIA-NN (free) software respectively.