Isotonitazene is a highly potent synthetic opioid in the benzimidazole class, associated with severe respiratory depression and overdose risk. In clinical and forensic toxicology, its presence can indicate illicit opioid exposure and may be missed by standard immunoassays, making confirmatory testing essential. Detection and quantitation are best achieved with LC-MS/MS or GC-MS workflows, often supplemented by high-resolution mass spectrometry for definitive identification.