Lipid family is regarded as vital metabolites in energy storage, cellular membrane construction as well as in signaling. Its structural diversity, thereby the complexity in physical/chemical property, poses analytical challenge for a structural-specific, comprehensive and quantitative analysis for a large spectrum of lipids. Currently, lipidomics tool based on the state-of-the art mass spectrometric technology, mainly shotgun lipidomics and liquid chromatography-mass spectrometry (LC-MS)–based lipidomics greatly paves the path to a better knowledge for lipid metabolism. Nevertheless, several aspects are still hindering the analytical capacity. On one hand, traditional extraction procedures could not satisfy multi-platform measurements, including lipidomics, in the case of limited sample amount. On the other hand, peak capacity in conventional LC-MS lipidomics is limited, especially for lipidomics analysis in extremely complex samples. In addition, most lipidomics merely focus on “snapshots” rather than capture the dynamics of lipid metabolism of individual lipid species on a network scale. Accordingly, we developed novel strategies to cope with above-mentioned limitations. A simultaneous extraction method of metabolome and lipidome was developed using methyl tert-butyl ether. With one single extraction, targeted and non-targeted metabolomics as well as lipidomics were performed for a small amount of tissue (2.5 mg muscle). In the meantime a novel on-line two dimensional LC-MS method using stop-flow mode was established for lipid separation, a higher peak capacity was obtained without losing sensitivity which usually occurs in 2nd dimension due to dilution effect. Besides, a novel tool employing stable isotope-assisted lipidomics combined with non-targeted isotopomer filtering was established, enabling to unravel the complex dynamics of lipid metabolism, 203 labeled lipids, corresponding to 692 isotopomers, and dynamic information were achieved in cells treated with [U-¹³C]-palmitate. Collectively, improvements in the lipidomics working pipeline including extraction, separation and dynamic metabolism dissection strategy have been made for a better lipidomics study.