Method development and optimization was performed for the quantification of tobacco specific nitrosoamines (TSNAs) in tobacco leaf. The objective was to shorten run time and compare the new method to the previous method. The evaluation of method performance indicated that the matrix effects, reproducibility, and detection limits were reduced. In the previous method, the initial stage of the ISO/CD TS 22304 sample preparation was performed with aqueous sodium hydroxide. Using the new method, samples were prepared using a single step extraction procedure with an ammonium acetate buffer as described in earlier collaborative studies. Quantification was performed using a new ultrahigh performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) method with electrospray ionization. The UHPLC method was optimized to separate the four common TSNAs using a gradient elution profile and a reversed-phase column. Following optimization, validation experiments were performed for the updated sample preparation and instrumental analysis procedures. The results from the validation of the newly developed method indicated that along with decreased run time, the method performance improved.The run time was reduced from 12 to 4.2 minutes. The matrix effects defined here as the degree of suppression was evaluated by comparing the response of each TSNA in the presence of tobacco matrix to the response in solution. The results illustrates that suppression was generally lower with the exception of NNN and NAT in oriental and NAB in both oriental and burley tobacco. The reproducibility of the method was reduced from up to 16% to less than 10% RSD. The limits of detection for the two methods were significantly lower as indicated by ANOVA, P=0.003.