Pantothenic acid (vitamin B₅) is found in a number of food groups. In food derived from living organisms, pantothenic acid primarily exists in its free form or in forms bound to coenzyme A or acyl carrier proteins. Free pantothenic acid is important from a nutritional perspective since the it is a dominant form and can be absorbed directly from the small intestine without going through the digestive system. This study aims to develop an accurate analytical method for free pantothenic acid using isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Beef was selected as a matrix due to a its higher pantothenic acid content compared to vegetables or grains. In order to increase extraction efficiency from a protein-rich matrix, papain (used as protease) was added to the extractant. However, the complicated clean-up process combining anion exchange and reversed-phase solid-phase extraction was required to reduce ionization suppression. And interfering peaks was observed near a retention time of the analyte during multi-reaction monitoring (MRM) detection. Therefore, it was determined to proceed extraction without papain. Single reversed-phase solid-phase extraction was enough for sample cleanup. Matrix effect profiles of sample extractants were obtained by using a post-column infusion system and no significant ion suppression was observed. Under the suggested extraction conditions, the influence on pantetheine hydrolysis was also investigated. It was revealed that the pantetheine was not converted to pantothenic acid. In a further study, a validation of developed method will be carried out.