Introductions: Psychoactive drugs represent a general term for substances with exceptionally powerful psychotropic effect, which were used as medications or more frequently for achieving certain sensation or state of mind during history. Lately, due to globalization and general technological advancements, we are faced with increasing number of new psychoactive substances, usually synthesized in laboratory. Because of insufficient data on pharmacological effect of these substances, they pose a serious threat for human health, especially among younger population more inclined to experimentation with such substances. Objective: The objective of this paper was to describe optimal method for detection and identification of novel psychoactive substances in patients' biological samples and to determine a trend of their consumption. Materials and methods: Materials, used in experimental section of this paper, were urine and blood serum of randomly picked patients which were prepared for toxicological analysis using extraction and derivatization. Method used for qualitative toxicological analysis was gas chromatography – mass spectrometry (GC – MS) which represents laboratory „gold standard“ method because of its accuracy, specify, sensitivity and precision. Results: Urine and blood serum samples are suitable biological material for qualitative analysis of new psychoactive substances because of standardization and ease of sampling. Analytical method of choice was gas chromatography with mass spectrometry. However, trends of consumption of new psychoactive substances should not be defined based on such analysis due to range of limiting factors from confined number of samples to violating the rule of representativeness. Key words: qualitative analysis, biological samples, novel psychoactive substances, gas chromatography, mass spectrometry