A versatile method for the analysis of TSNAs has been applied to different tobacco sample matrices such as tobacco filler, smokeless and raw tobacco materials, as well as cigarette smoke. The method used an HPLC separation on a Phenomenex Gemini C18 column with 3 micron particle size, and gradient mobile phase with aqueous ammonium acetate buffer/acetonitrile and acetic acid/acetonitrile as mobile phases A and B. The detection was performed by MS/MS with multiple reaction monitoring (MRM) in positive mode using specific transitions precursor ion to product ion, specific for each TSNA compound. The instrumentation used for the analysis was a 1290 Infinity from Agilent Technologies and API 4000 from AB Sciex. The method provides good selectivity with no interference from the sample matrix. For this reason, after the sample extraction, no clean up procedure was necessary regardless of the sample type. The method has a wide calibration range (1-600 ng/mL for NNN, NAT and NNK, 0.25-150 ng/mL for NAB) that allows the analysis to be performed on all tobacco and smoke samples without modification. The method is rapid, highly reliable, and shows excellent repeatability and robustness. The procedure has been applied successfully in the laboratory for a number of years and on a variety of samples.