Novel analytical platforms for high-throughput determination of lipid turnover in vivo have been developed based on partial metabolic 2H2O labeling. The performance on lipid kinetics measurement of our methods was validated in three different liquid chromatography-mass spectrometry (LC-MS) setups: MS-only, untargeted MS/MS, and targeted MS/MS. The MS-only scheme consisted of multiple LC-MS runs for quantification of lipid mass isotopomers and an extra LC-MS/MS run for lipid identification. The untargeted MS/MS format utilized multiple data-dependent LC-MS/MS runs for both quantification of lipid mass isotopomers and lipid identification. An in-house software was also developed to streamline the data processing from peak area quantification of mass isotopomers to exponential curve fitting for extracting the turnover rate constant. With HeLa cells cultured in 5% 2H2O media for 48 h, we could deduce the species-level turnover rates of 108 and 94 lipids in the MS-only and untargeted MS/MS schemes, respectively, which covers 13 different subclasses and spans 3 orders of magnitude. Furthermore, the targeted MS/MS setup, which performs scheduled LC-MS/MS experiments for some targeted lipids, enabled differential measurement between the turnover rates of the head and tail groups of lipid. The reproducibility of our lipid kinetics measurement was also demonstrated with lipids that commonly detected in both positive and negative ion modes or in two different adduct forms.