The gut microbiota supports a number of beneficial actions in our body. For example, it provides protection against pathogens, contributes to the maturation of the immune system, and regulates host metabolism. The composition of the gut microbiota is influenced by people's living conditions, various diets, and antibiotics. Changes in the composition of the gut microbiome have been observed in many diseases, including obesity, cardiovascular disease, inflammatory bowel disease, diabetes and several cancers. Among them, short-chain fatty acids (SCFAs) are the major metabolites produced from the fermentation of dietary fiber by these gut microbes. Most SCFAs can be identified through the gut, but it is difficult to analyze the obtained SCFAs in the same amount due to biodiversity problems including the water content of the gut. In addition, through recent studies, it was known that the SCFAs obtained from plasma and serum also differ by group. Here, we present an LC-MS/MS technique for the analysis of SCFAs using serum. This technology enables fast and accurate analysis based on scheduled MRM (sMRM) analysis based on sample derivatization and LC-MS, including fast and simple sample preparation and short LC runtime. As a result, it was possible to confirm the qualitative and quantification of acetate, butyrate, isobutyrate, acetylate and valerate.