Electrospray ionization mass spectrometry (ESI-MS) is an efficient soft ionization procedure for macro biomolecules. However, it is a rather delicate process to produce charged molecules for mass-to-charge ratio (m/z) based measurement. In this chapter, the mechanism of ESI is briefly presented, and the experimental pipeline for quantitative profiling of plasma proteins (prefractionation immunodepletion, protein isotope tagging, 2D-HPLC separation of intact proteins, and LC-MS) is presented as applied by our group in studies of cancer biomarker discovery.