Bioanalysis of new psychoactive substances (NPS) is very challenging due to the growing number of compounds with new chemical structures found on the drugs of abuse market. Screening, identification, and quantification in biosamples are needed in clinical and forensic toxicology settings, and these procedures are more challenging than the analysis of seized drug material because of extremely low concentrations encountered in biofluids but also due to diverse metabolic alterations of the parent compounds. This article focuses on bioanalytical single- and multi-analyte procedures applicable to a broad variety of NPS in various biomatrices, such as blood, urine, oral fluid, or hair. Sample preparation, instrumentation, detection modes, and data evaluation are discussed as well as corresponding pitfalls. PubMed-listed and English-written original research papers and review articles published online between 01 October 2012 and 30 September 2017 were considered.