In the last years, a global awareness has arisen from the reported harmful effects and public health risks associated with the consumption of new psychoactive substances (NPSs). Improving efforts in the detection and identification of these substances have emerged as a global analytical challenge involving the large range of NPSs' chemical structures and the variety of conventional and non-conventional biological matrices. Indeed, detection capabilities and screening tools impact many fields and settings, including seized products analysis, workplace and roadside drug controls, emergency rooms, drug addiction treatment clinics, post-mortem and criminal caseworks, law enforcement and health interventions. Colorimetric, immunochemical and chromatographic-mass spectrometry techniques have been investigated and developed for the rapid identification of NPSs. Considering the continuous emergence of new substances, this review offers a panoramic view on the current status of analytical approaches for the rapid screening of NPSs, including, when available, data on conventional and non-conventional biological matrices. Although some of the presented methods are sound and promising, their applications are still limited, thus proving the importance of further investigations. New screening and sensitive targeted methods for NPS and their metabolites should be developed in different types of biological matrices, where concentration of substances and matrix effects can be significantly different.