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The role of lipids in human health and disease is taking the center stage. In the last decades, there has been an intense effort to develop suitable methodologies to discover, identify, and quantitatively monitor lipids in biological systems. Recent advancement of mass spectrometry technology has provided a variety of tools for global study of the lipid “Omes”, including the quantification of known lipid molecular species and the identification of novel lipids that possess pathophysiological functions. Lipidomics has thus emerged as a discipline for comprehensively illuminating lipids, lipidderived mediators and lipid networks in body fluids, tissues and cells. However, owing to the complexity and diversity of the lipidome, lipid research is challenging. Here, the experimental strategies for lipid isolation and characterization will be presented, especially for those who are new to the field of lipid research. Because lipids are known to participate in a host of protein signaling and trafficking pathways, the review emphasizes the understanding of interactions between cellular components, in particular the lipid-protein interrelationships. Novel tools for probing lipid-protein interactions by advanced mass spectrometric techniques will be discussed. It is expected that by integrating the approaches of lipidomics, transcriptomics and proteomics, a clear understanding of the complex functions of lipids will eventually be translated into human diseases.