Exposure to smokeless tobacco (ST) has been reported to elicit diverse biological effects at the cellular level. Currently, there is no consensus regarding parameters that will impact cellular response. Various conditions have been described for 1) preparation methods of ST extracts 2) dosage in cell culture or 3) detailed analyses of the ST preparation that is used for the treatment of cells. Hence, we have evaluated methods for preparation and analysis of ST extracts using 2S3 reference smokeless tobacco. Ten percent (w/v) preparations of ST were extracted in DMSO (ST/DMSO) or complete artificial saliva with enzymes (ST/CAS) for up to 24 h, and analyzed for the presence of nicotine, 21 polycyclic aromatic hydrocarbons (PAHs) and four tobacco specific nitrosamines (TSNAs). Nicotine extraction efficiency was ~90% under all conditions. While PAHs were extracted equally in DMSO at 2 vs. 24 hours, their extraction was found to be generally higher in DMSO compared to ST/CAS. TSNA extraction efficiency was equivalent for both 2 and 24 h ST/DMSO. However, TSNAs were significantly elevated in the 24 h ST/CAS-extracted sample, compared to the 2 h ST/CAS-extracted sample. We hypothesize that the increase in TSNA levels in the 24 h ST/CAS extractions is artifactual due to microbial action occurring during sample preparation. A time course of ST/CAS extracts prepared with and without antibiotics was performed. Nicotine and PAH levels were unaltered by the presence of antibiotics. Significantly, antibiotics prevented increases over time in the levels of microbes, TSNAs and nitrite/nitrate ratio. Thus, for extraction times greater than 2 h in physiologically relevant aqueous buffers such as ST/CAS, consideration must be given to potential microbial-driven artifact formation (e.g. TSNAs) which may skew biological assay results. When conducting extractions longer than 2 h, the addition of an antibiotic, or other appropriate mitigant is recommended, as necessary, to minimize artifact formation.