Varenicline (VRC) is one of the most widely used and effective drugs used to treat smoking cessation. Nevertheless, amplifies the likelihood of suicidal ideation in individuals who have mental illness or pre-existing psychiatric issues. Current studies on this subject also highlighted the risks including behavioral changes, depression, and violence. In addition to causing intoxication, VRC also induces behavioral toxicity, which can be utilized as a defense in criminal cases. This study presents a simple and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that utilizes liquid-liquid extraction to quantification of VRC in plasma samples. The method was validated according to the SWGTOX guidelines and tested on plasma samples of patients under smoking cessation treatment to determine its applicability. All samples in the study were collected with patients’ consent and in line with ethical guidelines. As a result, VRC was found in the range of 6.20–24.04ng/mL in the plasma samples of patients. It is believed that quantitation of VRC in biological samples such as plasma in a case will be of great use in forensic toxicology and also in understanding its impacts on behavioral changes.