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Conformational biosensors to monitor the activation state of G protein-coupled receptors are a useful addition to the molecular pharmacology assay toolbox to characterize ligand efficacy at the level of receptor proteins instead of downstream signaling. We recently reported the initial characterization of a NanoBRET-based conformational histamine H₃ receptor (H₃R) biosensor that allowed the detection of both (partial) agonism and inverse agonism on living cells in a microplate reader assay format upon stimulation with H₃R ligands. In the current study, we have further characterized this H₃R biosensor on intact cells by monitoring the effect of consecutive ligand injections in time and evaluating its compatibility with photopharmacological ligands that contain a light-sensitive azobenzene moiety for photo-switching. In addition, we have validated the H₃R biosensor in membrane preparations and found that observed potency values better correlated with binding affinity values that were measured in radioligand competition binding assays on membranes. Hence, the H₃R conformational biosensor in membranes might be a ready-to-use, high-throughput alternative for radioligand binding assays that in addition can also detect ligand efficacies with comparable values as the intact cell assay.