In recent years, high-throughput lipid profiling has contributed to understand the biological, physiological and pathological roles of lipids in living organisms. Across all kingdoms of life, important cell and systemic processes are mediated by lipids including compartmentalization, signaling and energy homeostasis. Despite important advances in liquid chromatography and mass spectrometry, sample extraction procedures remain a bottleneck in lipidomic studies, since the wide structural diversity of lipids imposes a constrain in the type and amount of lipids extracted. Differences in extraction yield across lipid classes can induce a bias on down-stream analysis and outcomes. This review aims to summarize current lipid extraction techniques used for untargeted and targeted studies based on mass spectrometry. Considerations, applications, and limitations of these techniques are discussed when used to extract lipids in complex biological matrices, such as tissues, biofluids, foods, and microorganisms.