Venlafaxine is a phenethylamine derivative widely prescribed for the treatment of depression which inhibits both serotonin and norepinephrine reuptake (SNRI). In treatment with antidepressants of patient with depression and other psychiatric disorders there is also increased risk of suicidal thought and behaviour. Several lethal intoxications involving venlafaxine usually among psychotic patients have been reported in the literature. Sample preparation is of the greatest significance for a successful toxicological analysis. The development of simple, effective and rapid extraction procedures of drugs from post-mortem biological samples is a challenge. Headspace-solid phase microextraction (HS-SPME) offers significant advantages such as simplicity, low cost, compatibility with analytical systems, automation and solvent-free extraction. The aim of our work was the optimization of a HS-SPME procedure for the determination of venlafaxine in post-mortem biological samples by gas chromatography (GC) with nitrogen-phosphorous detection (NPD). Venlafaxine was extracted on 100 μm Polydimethylsiloxone Coating-Red (PDMS) SPME fiber and determined by GC-NPD. Salt addition, extraction temperature, preheating and extraction time were optimized to enhance the recovery of the extraction from aqueous solution spiked with venlafaxine. Finally the developed procedure was applied to post-mortem biological samples of a fatally poisoned woman by venlafaxine. The drug was quantified in post-mortem blood gastric and oesophagus contents of the deceased woman. A simple and rapid procedure using HS-SPME was developed for sample preparation of venlafaxine in post-mortem biological samples prior to GC-NPD determination. Validation data was satisfactory, thus enabling application in the toxicological analysis of forensic samples.