Short-chain fatty acids are organic acids with 1 - 6 carbon atoms. Their physiological functions and clinical applications have attracted considerable attention. Their high polarity, low ultraviolet (UV) absorbance, low contents in biological samples, high volatility, good water-solubility and easy adsorption on metal and glass surfaces result in some difficulties in their separation and determination. This review focuses on the methods for extraction of short-chain fatty acids in biological samples such as feces, urine, blood and culture solutions by distillation, high speed centrifugation, liquid-solid extraction, solid phase microextration, supercritical fluid extraction and liquid phase microextraction and determination by gas chromatography, high performance liquid chromatography and capillary electrophoresis. Sixty-three references are cited.