This review critically examines recent strategies for lipid analysis, covering sample pretreatment, instrumental analysis, and data interpretation from 2019 to 2023. Lipids, crucial biomolecules involved in various physiological and pathological processes, are challenging to analyze due to their diverse stereochemical properties and compositions. The review discusses the advantages and disadvantages of different extraction methods, emphasizing the importance of proper sample preparation to ensure accurate and reliable lipid identification and quantification. Mass spectrometry (MS) is highlighted as the most widely used technique, capable of direct infusion or combined with separation techniques like liquid chromatography (LC) or gas chromatography (GC). Special attention is given to the evaluation and interpretation of data obtained from lipid analyses, with a focus on accurate, precise, robust, and reliable analytical strategies. The review also explores targeted and untargeted approaches, the use of derivatization techniques, and the integration of ion mobility spectrometry (IMS) and supercritical fluid chromatography (SFC) with MS. Finally, it provides an overview of computational tools and software for data processing, statistical analysis, and enrichment analysis in lipidomics.This review critically examines recent strategies for lipid analysis, covering sample pretreatment, instrumental analysis, and data interpretation from 2019 to 2023. Lipids, crucial biomolecules involved in various physiological and pathological processes, are challenging to analyze due to their diverse stereochemical properties and compositions. The review discusses the advantages and disadvantages of different extraction methods, emphasizing the importance of proper sample preparation to ensure accurate and reliable lipid identification and quantification. Mass spectrometry (MS) is highlighted as the most widely used technique, capable of direct infusion or combined with separation techniques like liquid chromatography (LC) or gas chromatography (GC). Special attention is given to the evaluation and interpretation of data obtained from lipid analyses, with a focus on accurate, precise, robust, and reliable analytical strategies. The review also explores targeted and untargeted approaches, the use of derivatization techniques, and the integration of ion mobility spectrometry (IMS) and supercritical fluid chromatography (SFC) with MS. Finally, it provides an overview of computational tools and software for data processing, statistical analysis, and enrichment analysis in lipidomics.