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An efficient separation and quanti�cation of the individual neutral and polar lipid classes and their constituent fatty acids was achieved by the combination of two different detection techniques: Iatroscan TLC-FID and GC-FID. e solvent composition and ratio of development system, the sample size, the �delity, and precision were tested in order to estimate the effectiveness of separation of individual neutral and polar lipid classes and the quantitative reproducibility of the Iatroscan TLC-FID technique. GC-FID method, with a high-quality capillary column, allowed sensitive and reproducible fatty acid qualitative and quantitative analyses, separation of fatty acid structural isomers (e.g., n-C16:0, iso-C16:0 and anteiso-C16:0), positional isomers (e.g., C18:1 -9 and C18:1 -7), geometrical isomers (cis-trans), and homologues (e.g., C16:0, C17:0, C18:0, etc.) in standards and complex lipid samples. Seventeen (17) lipid classes and ��y-two (52) saturated (SFA), monounsaturated (M�FA), and polyunsaturated (P�FA) fatty acids were identi�ed and quanti�ed, respectively, in samples of standard lipid and fatty acid mixtures, simulating the composition of natural lipids and their fatty acid methyl esters in common foods. e wide number of applications establishes this combination of Iatroscan TLC-FID and GC-FID methods as a powerful tool for lipid class and fatty acid analysis of any fat origin.