Smokeless tobacco manufacturers have been required to annually report concentrations of nicotine in smokeless tobacco products (STP) sold in the United States to the Centers for Disease Control and Prevention (CDC) since 1999. The CDC protocol requires nicotine measurements conducted by gas chromatography with a flame ionization detector (GC-FID). Since the CDC protocol specifies a non-selective detector, new matrices and matrices with known interferences often require that calibration be performed by standards addition to accurately determine nicotine concentrations. The objective of the work was to validate and compare a more rapid, sensitive and selective method using GC with mass spectrometry detection (GC-MS) to the CDC’s GC-FID protocol. Statistical analysis (e.g., Schuirmann’s two one-sided tests (TOST) approach) determined that the two methods can be considered equivalent for the quantitation of nicotine in STPs and are thus both suitable for regulatory reporting. The GC-MS method had a broader calibration range (0.8-50 mg/g) compared to the CDC protocol (3.0-60 mg/g) and had no matrix interferences due to the selectivity of MS detection. The GC-MS method demonstrated a more than a tenfold increase in sensitivity. This method was validated for the quantitative analysis of nicotine as well as 3 additional alkaloids (nornicotine, anabasine, anatabine) in STPs, leaf tobaccos, cigarette filler, pipe tobacco and cigar filler.