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This study describes a novel technique for direct analysis of thiocyanate in human saliva using negative electrospray-ion mobility spectrometry (ESI-IMS) and without any considerable sample pretreatment. The ESI-IMS system with nearly complete desolvation cannot be useful for salivary thiocyanate analysis because complete overlapping occurs between the format peak of the solvent and the thiocyanate peak. In addition, the chloride ions existing in saliva produce a very broad peak, which has a drastically interfering effect on SCN(-) determination. In this study, with a little change in the operation conditions of the apparatus (lowering the flow rate of the drift gas), it was possible to overcome these problems. The desolvation process was decreased in the lower flow of drift gas, and this caused the SCN(-) peak to be separated completely from other interference peaks such as chloride ions. The results obtained in this proposed methodology provide the detection limit of 0.003 microg/mL and the linear dynamic range from 0.01 to 1.00 microg/mL for the thiocyanate. Several human saliva samples were analyzed with the proposed negative ESI-IMS, and the satisfactory results confirm the capability of the method for direct analysis of salivary thiocyanate.