Venlafaxine is most commonly prescribed for major depression and/or anxiety disorders. Fatal Venlafaxine overdoses have been reported. Forensic identification of an analyte requires the use of at least two methods that employ different physical and chemical principles. In the present work, Colour Tests, TLC, UV-spectroscopy, HPLC with multi-wave detection have been used for detection and determination of Venlafaxine in the liver tissue. The effective method of Venlafaxine isolation from the biological sample by drug elution with chloroform from the tissue homogenized by its grinding with anhydrous sodium sulphate followed by the extraction clean-up procedure with the n-hexane-acetonitrile solvent system has been developed. Resolution of the method was 51.3 % (RSD=5.5 %). Quantitative determination was performed by HPLC at 280 nm over the concentration range of 23.6–400 mg/mL with accuracy of 100.0–102.7 % and precision of 0.2–11.2 % depending on the concentration level. The calibration curve was represented by the following regression Y=1.64·10-4X, LOQ=23.6 mg/mL (10SDa 2/b), LOD=15 μg/mL (S/N=3:1) for Venlafaxine in methanol solutions. It has been shown that the matrix co-eluting components do not interfere with the quantitative determination of Venlafaxine by HPLC at wavelength of 280 nm.