Novel synthetic opioids (NSO) are an emerging class belonging to new psychoactive substances (NPS). They include fentanyl (e.g., acetylfentanyl, ocfentanil, carfentanil, furanylfentanyl, fluorofentanyl) and non-fentanyl analogs (e.g., U-47000, AH-7921, MT-45). NSO include potent μ-opioid receptor agonists with low dose required to produce the desired effects leading to a high risk of fatal overdose. The use of these molecules is spreading rapidly among opioid users, and is the main cause of opioid overdose in the United States. In recent years, we have seen an increase in cases of NSO poisoning in Europe, including fatal intoxications. The use of NSO among opioid users is dangerous and represents a major public health issue. These molecules have pharmacological properties different from those of conventional opiates. In case of overdose, respiratory depression appears quickly and could be life-threatening. As for conventional opioids, the management of intoxicated patients is based on naloxone. However, the doses and duration of the treatment vary according to the pharmacodynamic and pharmacokinetic characteristics of the molecules. NSO addiction management is based on the same molecules as conventional opioids: methadone and buprenorphine. Detection and quantification in biological matrices rely on liquid chromatography/tandem mass spectrometry and gas chromatography/tandem mass spectrometry, given the pharmacological potency of many NSO, the quantities administered are small and the concentrations found in biological matrices are weak, requiring the development of sensitive methods. Given the rapid diversification of this group of NPS, it is essential to continuously increment the libraries. Novel approaches were recently developed and are promising to detect unknown compounds such as activity based bioassays.