The authors have characterized a set of cannabinoid CB₂ receptor ligands, including triaryl bis sulfone inverse agonists, in a cell-based receptor/β-arrestin interaction assay (DiscoveRx PathHunter^™). The results were compared with results using a competitive ligand binding assay, and with effects on forskolin-stimulated cAMP levels (PerkinElmer LANCE^™). The authors show good correlation between the 3 assay systems tested, with the β-arrestin protein complementation assay exhibiting a more robust signal than the cAMP assay for cannabinoid CB₂ agonists. Further assay validation shows that DiscoveRx PathHunter^™ HEK293 CB₂ β-arrestin assay can be carried out from cryopreserved cell suspensions, eliminating variations caused by the need for multiple cell pools during live cell screening campaigns. These results, and the authors' results evaluating a test set of random library compounds, validate the use of ligand-induced interaction between the human cannabinoid CB₂ receptor and β-arrestin as an appropriate and valuable screening platform for compounds specific for the cannabinoid CB₂ receptor. (Journal of Biomolecular Screening 2009:49-58)