Technical Abstract: Pantothenic acid, vitamin B5, is a B-family water-soluble vitamin. Its richest sources are yeast and animal organs (liver, kidney, heart, brain), but eggs, milk, vegetables, legumes, and wholegrain cereals are also common sources. Pantothenic acid is important in the oxidation of fatty acids and carbohydrates and the synthesis of amino acids, fatty acids, ketones, cholesterol, phospholipids, and steroid hormones. As part of acetyl CoA or acyl carrier protein, it is essential in energy metabolic pathways. Historically, pantothenic acid analysis has relied on chemical and physical methods, animal bioassay, and microbiological methods. These methods are generally outdated. Analysis of pantothenic acid in multi-vitamin dietary supplements using high-performance liquid chromatography (HPLC) and optical detectors (e.g. diode array detector or fluorescence detector) is difficult since pantothenic acid does not have a chromophore, nor does it fluorese. In this book chapter, analysis of pantothenic acid using modern HPLC mass spectrometry (LC/MS) has been discussed in detail. The preferred ionization technique, mass analyzer, mobile phase, HPLC elution program, and HPLC analytical columns have all been covered. An example is LC/MS method that is capable of simultaneous determination of multiple water soluble vitamins (WSV: niacin, thiamine, pyridoxine, riboflavin, folic acid, biotin, pantothenic acid and ascorbic acid) in dietary supplements is also given.