It is critical to characterize the release of nicotine and tobacco-specific nitrosamines (TSNA) from smokeless tobacco products for safety evaluation and product quality control. A novel model mouth system for evaluating the in vitro nicotine and TSNA release behavior of a smokeless tobacco product (moist snuff) has been developed. The system consists of the release medium reservoir module, the flow speed control module, the temperature control module, nicotine release module, and release solution collection module, and simulates buccal condition in terms of temperature, saliva compositions, and the rate of saliva production, etc. Nicotine concentration was determined with a reversed-phase high-performance liquid chromatographic (RP-HPLC) method. TSNA concentration was determined using a new Liquid chromatography-electrospray tandem mass spectrometry (LC-ESI MS/MS) method. The influences of flow rate and temperature of the release medium on the nicotine release rate were studied. The performance of the model mouth system was compared with in vivo data of nicotine and TSNA release in human volunteers. The model mouth system was applied to evaluate nicotine and TSNA release in 21 brands of commercially available moist snuff, and the effects of product weights, nicotine concentration, and product pH values on nicotine and TSNA release were evaluated. The model mouth system and the analytical method were shown to be a useful tool in assessing the bioavailability of nicotine and TSNA to moist snuff users.