The worldwide spread of novel psychoactive substances (NPS) in the illicit drug market and their continuous increase in number and type, for the purpose of bypassing controlled substance legislation, represents a continuing challenge for forensic scientists, clinicians and enforcement authorities. We aim to provide information regarding the most urgent harms related to NPS consumption in different world regions and the current state of the art for NPS analysis. Unfortunately, the identification of NPS in biological samples is controversial, especially when samples are limited, or the drug is promptly and extensively metabolized. This causes a lack of information on their real diffusion in different parts of the world and in different populations. New technologies and instrumental detection of NPS in alternative samples are offering comprehensive information about NPS use. The lack of detection and underreporting of NPS in biological samples makes it difficult to obtain complete qualitative and quantitative information about NPS prevalence. The most innovative strategies that have been proposed in the last 2 years to assist NPS analysis and possibly facilitate the understanding of the NPS diffusion around the world are presented.