Recent Analytical Methodologies in Lipid Analysis reviews the latest strategies for lipid analysis from sample pretreatment to instrumental analysis and data interpretation over the past five years. Lipids are essential biomolecules involved in various cellular processes, and their imbalance can lead to diseases such as diabetes, chronic inflammation, and neurodegenerative disorders. Due to the complexity of lipid structures and their diverse stereochemical properties, lipid analysis is challenging. This review discusses various extraction methods, instrumental techniques like mass spectrometry (MS), and data analysis approaches used in lipidomics. Sample pretreatment is crucial in lipid analysis, as lipids are prone to oxidation and hydrolysis. Common extraction methods include single organic solvent extraction (SOSE), one-phase extraction (OPE), and liquid-liquid extraction (LLE). OPE is gaining popularity due to its simplicity and effectiveness in extracting less polar lipids. Other methods, such as solid-phase extraction (SPE) and solid-phase microextraction (SPME), are also used for lipid analysis. Derivatization is often used in GC analysis to enhance ionization efficiency and selectivity. Instrumental analysis of lipids typically involves mass spectrometry, which is the most widely used technique. Direct infusion MS allows for rapid and sensitive analysis, while liquid chromatography (LC) combined with MS provides higher resolution and separation capabilities. Techniques like tandem mass spectrometry (MS/MS) and ion mobility spectrometry (IMS-MS) are used for lipid identification and quantification. Additionally, mass spectrometry imaging (MSI) enables the visualization of lipid distribution in tissues and cells. Data analysis in lipidomics involves processing raw data, statistical analysis, and enrichment analysis. Tools like Skyline, MSDial, and LipidSearch are used for data interpretation. The review highlights the importance of accurate and reliable analytical methods in lipidomics, which can contribute to understanding diseases at the molecular level and identifying potential biomarkers or therapeutic targets.Recent Analytical Methodologies in Lipid Analysis reviews the latest strategies for lipid analysis from sample pretreatment to instrumental analysis and data interpretation over the past five years. Lipids are essential biomolecules involved in various cellular processes, and their imbalance can lead to diseases such as diabetes, chronic inflammation, and neurodegenerative disorders. Due to the complexity of lipid structures and their diverse stereochemical properties, lipid analysis is challenging. This review discusses various extraction methods, instrumental techniques like mass spectrometry (MS), and data analysis approaches used in lipidomics. Sample pretreatment is crucial in lipid analysis, as lipids are prone to oxidation and hydrolysis. Common extraction methods include single organic solvent extraction (SOSE), one-phase extraction (OPE), and liquid-liquid extraction (LLE). OPE is gaining popularity due to its simplicity and effectiveness in extracting less polar lipids. Other methods, such as solid-phase extraction (SPE) and solid-phase microextraction (SPME), are also used for lipid analysis. Derivatization is often used in GC analysis to enhance ionization efficiency and selectivity. Instrumental analysis of lipids typically involves mass spectrometry, which is the most widely used technique. Direct infusion MS allows for rapid and sensitive analysis, while liquid chromatography (LC) combined with MS provides higher resolution and separation capabilities. Techniques like tandem mass spectrometry (MS/MS) and ion mobility spectrometry (IMS-MS) are used for lipid identification and quantification. Additionally, mass spectrometry imaging (MSI) enables the visualization of lipid distribution in tissues and cells. Data analysis in lipidomics involves processing raw data, statistical analysis, and enrichment analysis. Tools like Skyline, MSDial, and LipidSearch are used for data interpretation. The review highlights the importance of accurate and reliable analytical methods in lipidomics, which can contribute to understanding diseases at the molecular level and identifying potential biomarkers or therapeutic targets.