G protein-coupled receptors (GPCRs) act as a signaling hub through G proteins and arrestins upon ligand binding. Biased ligands with pathway-selective activity have attracted much attention as drugs with lower side effects. However, it is yet to be clear how the multiple signaling pathways of GPCR are spatial-temporally regulated in living cell membrane. Here, we first demonstrate that the average diffusion coefficient of GPCR molecules in living cell membrane generally decreased upon activation. Then, we show that drug effects on multiple signaling pathways downstream of S1PR1, a model class A GPCR, can be estimated simultaneously from the diffusion dynamics of receptor molecules in a single cell by single-molecule time-lapse imaging. We also show a novel signalosome that regulates the signal-bias of the angiotensin signaling by AT1R, another class A GPCR. The NanoBiT assays and BRET imaging revealed the AT1R/G protein/GRK preassembly complex in living cell membrane. The dual color single-molecule imaging analysis suggested that the preassembly complex regulates the signal bias in a confined region of the plasma membrane.