Venlafaxine (VEN), which was introduced into the therapy in 1990s, is one of the most often used antidepressants. The monitoring of its level in the human organism is recommended, particularly in the case of a patient suffering from other illnesses and treated with different drug substances interfering with VEN. The most popular diagnostic material for the quantitation of VEN is blood. The present study is the review on the methods of determination of VEN and its metabolites in blood and other human diagnostic materials, such as saliva and urine, and also in animal tissues. The paper also sums up the methods of extraction, i.e. liquid-liquid extraction, solid-phase extraction as well as modifications of these methods, such as liquid-phase microextraction and cloud-point extraction. According to the literature, it can be stated that the most popular method for the determination of VEN level and its main metabolite, O-demethylvenlafaxine, is HPLC with the UV or spectrofluorimetric detectors. Another separation method used for the determination of VEN is LC with MS. As a stationary phase, the most frequently the C18 column is applied and a mixture of acetonitrile with a phosphate buffer. For the determination of venlafaxine enantiomers, a chiral stationary phase was used.