The alkylindole (AI), WIN55212-2, modulates the activity of several proteins, including cannabinoid receptors 1 and 2 (CB1R, CB2R), and at least additional G protein-coupled receptor (GPCR) that remains uncharacterized with respect to its molecular identity and pharmacological profile. Evidence suggests that such AI-sensitive GPCRs are expressed by the human kidney cell line HEK293. We synthesized fourteen novel AI analogues and evaluated their activities at AI-sensitive GPCRs using [35S]GTPγS and [3H]WIN55212-2 binding in HEK293 cell membranes, and performed in silico pharmacophore modeling to identify characteristics that favor binding to AI-sensitive GPCRs versus CB1R/CB2R. Compounds 10 and 12 stimulated [35S]GTPγS binding (EC50s = 3.5 and 1.1 nM, respectively), and this response was pertussis toxin-sensitive, indicating that AI-sensitive GPCRs couple to Gi/o proteins. Five AI analogues reliably distinguished two binding sites that correspond to the high and low affinity state of AI-sensitive GPCRs coupled or not to G proteins. In silico pharmacophore modeling suggest 3 characteristics that favor binding to AI-sensitive GPCRs versus CB1R/CB2R: 1) an s-cis orientation of the two aromatic rings in AI analogues, 2) a narrow dihedral angle between the carbonyl group and the indole ring plane [i.e., O-C(carbonyl)-C3-C2] and 3) the presence of a carbonyl oxygen. The substituted alkylindoles reported here represent novel chemical tools to study AI-sensitive GPCRs.