As the tobacco industry continues to emphasize tobacco harm reduction, an array of innovative potentially reduced-risk products have emerged as alternatives to smoking cigarettes. These innovative products include a wide range of smoke-free products with different matrix types. This has resulted in the need for developing and validating multiple matrix-dependent analytical methods for the determination of nicotine, which can only be applied to specific matrix types. In this study, we developed and validated a novel method for the extraction and accurate quantitation of nicotine from a variety of commercially available tobacco products with differing matrices (e.g., pouched and loose traditional smokeless tobacco, lozenge, gum, nicotine pouch, and e-liquids). The method employed a modified QuEChERs extraction technique consisting of a liquid-liquid extraction using sodium hydroxide and acetonitrile in conjunction with UPLC-MS/MS using isotopically labeled internal standard, nicotine-methyl-d₃. The method was validated according to the International Council on Harmonization (ICH) guidelines and guidance from the Center for Tobacco Products (CTP) at the Food and Drug Administration (FDA) on validation of analytical methods used for tobacco products. For all sample types evaluated in the validation study, the recoveries using isotopically labeled internal standard were found to be within 89 to 109 % for fortification levels that were based on the inherent nicotine concentration for each sample matrix. Intra-day and inter-day method precisions were both determined to be ≤ 7 % relative standard deviation (%RSD). This analytical method has the advantage to reduce extraction and analysis time, is easy to implement and maintain, and is applicable to a wide range of smoke-free tobacco products. We believe this method has potential for standardization.