The analysis of low concentration water soluble vitamins in dietary supplements can be challenging with traditional UV detection. Some of the vitamins are poor UV absorbers and their accurate quantitation in highly complex samples especially when present at very low levels can lead erroneous results. Addressing these challenges requires use of modern analytical technologies such as UPLC-UV-MS to ensure methods are robust, reliable, sensitive and provide a high degree of confidence in the identity and quantity of measured compounds. The use of smaller-particle size UPLC columns can provide significant improvements in separation quality for all samples, however this is especially useful when analyzing multi-ingredient finished products. Dual-detection systems (LC-UV-MS) are also increasingly finding a home in QC laboratories where they can help to provide greater confidence in the identity and purity of measured peaks. This application demonstrates the benefits of UPLC-UV-MS technologies for accurate measurement of challenging vitamins in dietary supplements.