The aim of the study was to estimate the thiocyanate levels in saliva of cigarette smokers in comparison to e-cigarette smokers and nonsmokers. To improve our understanding of the influence of smoking on the oral level of thiocyanate, we conducted an assessment of human saliva, in 24 individuals (eight tobacco smokers, eight e-cigarette smokers, and eight nonsmokers). High-Performance Liquid Chromatography with ultraviolet detection (HPLC-UV) using a unique phosphatidylcholine column was applied in this assay. Thiocyanate ion was detected directly by its absorbance at 210 nm. The method presents a new application of the IAM (Immobilized Artificial Membrane) column for quantification of inorganic anions. The whole process meets the criteria of green chemistry because it was carried out without the use of organic solvents. For compensating matrix effects, an eight-point standard addition protocol was used to quantify the thiocyanate level in saliva samples. The calibration graphs were linear in the range of 5–100 mg L−1 with a correlation coefficient higher than 0.99. The thiocyanate concentrations in the saliva of tobacco smokers, e-cigarette smokers, and nonsmokers were found in the range of 121.25–187.54 mg L−1, 121.24–244.11 mg L−1, 33.03–79.49 mg L−1, respectively. The present study indicates an obvious statistically significant elevation in salivary thiocyanate level in tobacco smokers in comparison to nonsmokers. The phosphatidylcholine-based stationary phase proved to be suitable for the detection and quantification of the thiocyanate ion. The salivary thiocyanate levels in e-cigarette smokers were not significantly different in comparison to tobacco smokers but higher if compared to nonsmokers. The criterion for statistical significance was p < 0.05.