The global opioid crisis is a public health emergency characterized by the widespread misuse of opioid drugs, leading to high rates of addiction, overdose, and death. Initially driven by the over-prescription of opioid painkillers, it has evolved into a worldwide epidemic exacerbated by the rise of potent synthetic opioids. Benzimidazole opioids ('nitazenes') are one group within this class of compounds that have garnered increasing attention in recent years due to their addictive potential and growing presence in illicit drug markets. Because of their high potency, low concentrations in biological samples are expected, implying the need for very sensitive and selective methods. In the presented paper, an UHPLC-QqQ-MS/MS method was developed for the determination of 26 nitazenes in biological samples (including metabolites), with simultaneous separation of structural isomers. Biological samples were prepared using liquid-liquid extraction (LLE) at pH 9, and quantification was performed using MRM mode. Metonitazene-d