Modern oral nicotine products are becoming increasingly popular alternatives to smoking however the palatability is largely related to added flavorants which may have high chemical diversity. Furthermore, in clinical studies the measurement of remnant flavors in used products and in saliva during use are often critical for understanding the bioavailability of product components and the overall expected usage timeframe. In this study, a comprehensive strategy was developed and validated to quantify two non-volatile artificial sweeteners plus fourteen volatile flavor components in unused and used smokeless pouch products as well as in saliva for application in support of clinical trials. Analysis of artificial sweeteners required two independent LC-ESI-MS/MS injections using reversed phase and HILIC separation prepared from a single aqueous extract. The volatile flavor components were analyzed by GC-MS using an ethanol-based extract. Each method developed in this study demonstrates analytical characteristics well suited for clinical trial use in both matrices including low saliva volume requirements, low or sub-ppm limits of quantification, as well as good specificity and linearity. The development process, methodology, and exemplary validation results will be discussed.