Lipids are structurally diverse and functionally critical biomolecules playing key roles in energy storage, signal transduction, and membrane formation. Their precise regulation is essential for cellular homeostasis, with metabolic disruptions closely linked to conditions like atherosclerosis and diabetes, while lipid metabolic reprogramming has emerged as a hallmark of cancer. This review outlines recent advances in applying stable isotopes for lipid metabolic tracing, lipidomics analysis, and flux quantification. It discusses various labeling strategies and reviews dynamic characterization models such as mass isotopomer distribution (MID), carbon-average enrichment, isotopomer spectral analysis (ISA), and 13C-nonstationary metabolic flux analysis (13C-NMFA). The applications of isotope tracing in baseline metabolism, disease-induced reprogramming, and pharmacology are examined, alongside advancements in mass spectrometry imaging and single-cell lipidomics. Ultimately, stable isotope tracing offers a dynamic perspective for lipid metabolism, enabling the tracking of real-time pathway changes and metabolic fluxes. Combined with advanced mass spectrometry, this approach should significantly expand the understanding of physiology and disease mechanisms, highlighting its potential in pharmacological research and future developments in multimodal data integration and AI-driven spatiotemporal modeling.
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