Detailed assessment of the biotransformation of compounds and the activity of their metabolites is an extremely important element in the safety evaluation of a substance, both in preclinical and clinical studies. It should be noted that the metabolite may differ from the parent compound in terms of physicochemical properties and consequently pharmacological and toxicological properties. The purpose of the biotransformation of xenobiotics is to increase their hydrophilicity, which allows excretion in the urine. the metabolites of biotransformation phase I show undesirable pharmacological or toxic effects much more often. according to U.S. Food and Drug administration (FDA) guidance, if a metabolite in the human body accounts for more than 10% of the total amount of metabolites, its safety should be thoroughly assessed. Metabolites can interact more strongly or weakly, or to the same degree, with the same or a different molecular target as the parent compound. they can also display non-specific effects by, for example, damaging macromolecules (e.g. proteins, nucleic acids) in the way that free radicals do. In the case of new psychoactive substances (NPSs), the pharmacological properties, including metabolism, are largely unknown until they are traded illegally and their users begin to appear in departments of toxicology or forensic medicine. It is in this way that the activity profile and probable metabolic pathways of NPSs are determined. to determine the metabolites of NPSs is also an important toxicological skill in the forensic testing of biological samples (blood/urine/tissue) collected from victims, in which metabolites, not parent compounds, are usually found. Using examples from the authors’ research and the available literature, the article aims to present alternative methods of metabolism testing for NPSs. In vitro methods (application of microsomes, S9 fraction, hepatocytes, cytosol) are discussed and comparisons are made between the results of in vivo tests on animals and analyses of autopsy material. The experiments and the literature review demonstrate that by using in vitro methods the metabolism of NPSs can be predicted with high probability. By improving existing methods of metabolism research and creating new and alternative ones it will be possible to better understand metabolic pathways and better identify the NPS metabolites formed in the human body. This will contribute not only to the development of better methods of treating NPS poisoning, but will also be of use when compiling forensic and medical reports for the judiciary.