Amino acids are widely distributed in biological fluids and involved in many biological processes, such as the synthesis of proteins, fatty acids, and ketone bodies. The altered levels of amino acids in biological fluids have been found to be closely related to several diseases, such as type 2 diabetes, kidney disease, liver disease, and cancer. Therefore, the development of analytical methods to measure amino acid concentrations in biological samples can contribute to research on the physiological actions of amino acids and the prediction, diagnosis and understanding of diseases. This review describes the analytical methods reported in 2012–2016 that utilized liquid chromatography and capillary electrophoresis coupled with ultraviolet, fluorescence, mass spectrometry, and electrochemical detection. Additionally, the relationship between amino acid concentrations and several diseases is also summarized.