A selective and sensitive method based on derivatization with 2-thiobarbituric acid and ultra-fast liquid chromatographic separation is described for the determination of malondialdehyde (MDA) in chicken liver, muscles and adipose tissue, and in lard and fish oil. Preparation of samples involves acid hydrolysis and derivatization. Separation is achieved using an Accucore C18-column (2.6 μm, Hydro-RP, 150×3.0 mm), an acetonitrile gradient in water, and detection at 530 nm. The results indicate that external calibration based on standard solutions of MDA may be used for measuring the MDA concentration in adipose tissue, lard and fish oil due to the absence of matrix effects. The MDA concentration in protein-rich biological samples should be calculated as the difference between the MDA concentration measured in MDA-spiked and unspiked samples of the same specimen and mass and the known concentration of the MDA spike. For liver or muscle samples, we suggest using external calibration based on standard solutions of MDA added to the same mass of liver or muscles. The proposed method is suitable for rapid and sensitive analysis of MDA in samples of animal origin or in plant oils. The method can also be suitable for routine evaluation of oxidative stress in animal tissues and oxidative stability of biological materials and animal products.