Fatty acid profile is one of the factors affecting health values. Besides the fatty acid composition, the ratios between saturated, monounsaturated, and polyunsaturated fatty acids (SFA, MUFA and PUFA, respectively) and trans fatty acids play an important role. In the examination of the fatty acid profile, the deficiency/high level of fatty acids plays an important role in the diagnosis and treatment of diseases caused by fatty acid oxidation disorders (FAO) and peroxisomal disorders. Fat deficiencies in the body are usually caused by inadequate dietary lipid intake due to unbalanced nutrition, prolonged parenteral nutrition, or intestinal malabsorption. Major clinical manifestations associated with FAO include hypoketotic hypoglycemia, liver disease and failure, skeletal myopathy, dilated/hypertrophic cardiomyopathy, and sudden death. Disease-specific characteristic patterns of metabolites resulting from FAO disorders can be detected in blood, bile, urine, and cultured fibroblasts of many living and dying individuals. Quantitative determination of C8-C18 fatty acids is an important element of the study and differential diagnosis of candidate patients. With the fatty acid profile, patients can detect abnormalities when they are asymptomatic and under diet therapy. In peroxisomal disorders, increased serum levels of very long chain fatty acids (VLCFA, C22:0, C24:0, C26:0), pristanic and phytanic acids indicate Zellweger syndrome.