Quantification of proteins is one of the fundamental applications of proteomics, and has the potential to yield insights into the biology of organisms, especially the response to changes in the internal or external environment. Mass spectrometry-based protein quantification (label-based and label-free) has become a widely used approach to measure relative and absolute abundances of proteins, and has been applied for the discovery of disease-specific biomarkers for early diagnosis and prognosis . In this study, the generalized protocol for the quantification of SWATH-MS data is presented using Skyline software . SWATH-MS (Sequential Window Acquisition of all THeoretical fragment ion spectra coupled to Tandem Mass Spectrometry) is a novel quantification technique that operates in data-independent acquisition (DIA) mode and helps in recording all the peptides’ precursors present in the biological sample, including low abundant proteins (such as, plasma proteins). In this protocol, the key steps of quantitative analysis, such as ion chromatogram extraction and, quantification at peptide and protein levels have been presented using publically available dataset. The expression levels of peptides and proteins have been statistically evaluated to identify up and down-regulated proteins. This protocol has the potential to be used to measure the abundances and expressions of peptides extracted from different cancerous samples for the discovery of novel biomarkers. However, such an application shall be undertaken in a future study.