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Nitazenes represent a new synthetic opioids sub-class belonging to new psychoactive substances (NPSs). Their high pharmacological potency has led to numerous intoxications and fatalities, even at minimum doses. The aim of this study was to assess the stability of four nitazenes (etazene, flunitazene, isotonitazene and protonitazene) in dried blood spot (DBS) samples at different storage temperatures (room temperature and 4 °C) and determine the optimal storage conditions. Moreover, we developed and validated a new and fast liquid chromatography–high-resolution mass spectrometry method by the optimization of chromatographic conditions with the use of a different chromatographic column and mobile phases. Two concentrations, 1 and 5 ng/mL, were chosen based on the available data on nitazenes-related intoxications and their stability was evaluated at days 0 (control), 1, 7 and 30. The results showed that all analytes at 1 ng/mL were not detectable after 30 days at room temperature; a similar pattern was observed for 1 ng/mL etazene and isotonitazene samples when stored at 4 °C, whereas flunitazene and protonitazene decreased to a mean of 66% and 69% initial concentrations, respectively, at day 30. Differently, all analytes at 5 ng/mL were quantified above 44% and 41% initial concentrations at room temperature and 4 °C, respectively, showing a higher stability. The study of nitazenes stability in DBSs represents an important tool to determine the optimal sample storage conditions, such as temperature and time between sample collection and analysis. In contrast to another study, our study showed distinct stability behaviors for every investigated analyte, which also depended on the concentration. Therefore, it is difficult to define an optimal storage condition acceptable for all nitazenes. Room temperature proved to be the best medium- and long-term storage conditions for the highest concentrations, but the stability of low levels of flunitazene and protonitazene improved at 4 °C. [ABSTRACT FROM AUTHOR]