Recent years have witnessed a growing in interest in psychoactive substances, particularly those available in e-commerce. These have led to an increase in the number of drug-related poisonings, deaths, and road accidents. Illegal drugs are available on an unprecedented scale and cause previously unknown adverse effects, which creates a challenge for analysts to find rapid methods for identifying these substances and taking appropriate action in the shortest possible time. New psychoactive substances (NPSs) can be lethal at very low concentrations, which give particularly serious cause for concern. These drugs are easily accessible and often regarded (or claimed) to be safe, which encourages many people, in particular young people, to try them. The widespread use of these substances is compounded by the awareness that they are difficult to detect with the existing rapid screening tests. Simple, fast, sensitive, and specific methods for determining the largest possible number of black-market psychoactive substances and their metabolites are therefore essential. Such methods will facilitate treatment and increase the effectiveness of measures aiming to reduce drug addiction. The objective of this review article was to critically compare the most commonly used analytical methods for determining NPS and their metabolites in biological material, with special emphasis on the sample preparation process, and to highlight the possibilities offered by the existing analytical methods.