As the building blocks of proteins, amino acids support the diversity of life. They also exist in the body as individual molecules and individually play critical roles in many biochemical pathways. This chapter briefly discusses how the general structures of amino acids make them important to various biochemistries. It presents some examples where measurement of amino acids gives insight into the function of a biochemical pathway and may provide an indication of disease. Ultimately, the chapter examines analytical approaches to measure these molecules and offer guidance to ensure optimum assay characteristics such as accuracy, precision, and selectivity using methodologies involving LC-MS. Amino acids can also be funneled into metabolic pathways to be used as an energy source. Tyrosine is a precursor for dopamine, norepinephrine, and epinephrine; decarboxylation of glutamate and histidine results in gamma-aminobutyric acid (GABA) and hista-mine, respectively; and tryptophan can be metabolized to serotonin.