α-Lipoic acid (LA) is a cofactor functioning in four multienzymes: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, and the glycine cleavage enzyme systems. In each enzyme, LA is covalently bound to the ε-amino group of lysine residues. LA has been used as a medicine for treating diabetic condition in Europe and is also currently a popular supplement resource, and thus the metabolic fate after orally intake of LA attracts much attention whether externally administered LA is incorporated into protein bound form because large fraction of administered dose is not fully explained yet. Lipoamidase is the enzyme catalyzing formation and breakage of amide bond between LA and lysine ε-amino groups in the protein and thus is expected to play critical role in LA metabolism and function. In order to know how the enzyme lipoamidase is involved in the fate of LA in the body, a simple assay method is requested for determining lipoamidase activity in tissues. The method using simple HPLC detection of newly synthesized fluorescent substrate, dansyl-α-lipoyllysine, is discussed together with other previously reported methods.