<p>Synthetic cannabinoid receptor agonists (SCRAs) are marketed as ‘legal’ cannabis alternatives, but are often much more potent and toxic, increasing the risk of (severe) intoxication. Initially sold as herbal preparations, SCRAs are now increasingly found in e-liquids due to the growing popularity of e-cigarettes. Traditional analytical methods, such as (high-resolution) mass spectrometry, can detect these substances but face limitations regarding time and cost, and require sophisticated equipment and frequently updated mass spectral libraries. Additionally, the continuous emergence of new SCRAs, aiming at evading legislation or detection, further challenges these methods. Activity-based screening, evaluating a sample’s inherent cannabinoid activity rather than relying on structural identification, offers an effective alternative. Here, an in vitro CB₁/β-arrestin2 recruitment assay utilizing the NanoBiT^® principle was, for the first time, applied to an e-liquid from an intoxicated patient, demonstrating strong cannabinoid activity. Employing the assay to screen a set of 23 e-liquids identified six SCRA positive e-liquids. Moreover, in five e-liquids, a decreased CB₁ activity was observed and experimentally confirmed to be attributable to the presence of the natural cannabinoid cannabidiol (CBD). As this could potentially result in a false-negative screening, an adapted protocol was evaluated, incorporating the injection of a CB₁ agonist, CP55,940, while the assay was running. This improved methodology allowed the detection of both SCRAs and CBD in e-liquids. Furthermore, the fast and ‘untargeted’ nature of this approach makes it a future-proof method for the detection of SCRAs, serving as an effective first-line screening tool, complementing the conventional analytical techniques.</p>