Biomarkers, derived from carbon-based organic molecules and synthesized in terrestrial or aquatic environments, serve as indicators of biological processes. Their isotopic compositions offer insights into source partitioning and pathways, aiding paleoclimate studies. Lipid biomarkers, such as n-alkanes, fatty acids, hopanes, and steranes, provide compound-specific information which is essential for ecological research. Additionally, 3-hydroxy fatty acids (3-OH FAs) and glycerol dialkyl glycerol tetraethers (GDGTs) offer insights into temperature and pH in both terrestrial and marine ecosystems. Pretreatment procedures, solvent selection, and extraction techniques, such as Soxhlet extraction, ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and accelerated solvent extraction (ASE), offer advantages and limitations depending on the sample matrix and target compounds in lipids. Following extraction, lipids are separated using specific polarity-based solvents in short-column chromatography and subsequently analyzed in gas chromatography-mass spectrometer (GC-MS), liquid chromatography-mass spectrometer (LC-MS) for quantification of compounds, and GC coupled to isotope ratio mass spectrometer (IRMS) for carbon and hydrogen isotopic compositions of the respective compounds. The present chapter outlines comprehensive methodologies for the extraction, separation, and quantification of lipid biomarkers, where a detailed comparison of the advantages and disadvantages of sample pretreatment, solvent selection, and extraction techniques is discussed for successful lipid biomarker analysis in paleoclimate research.