G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gα i1, Gα i2 and Gα i3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gα i subunit, and cp173Venus fused to the Gγ 2 subunit as acceptor. The Gα i FRET biosensors constructs are expressed together with Gβ 1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gα i FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gα i FRET sensor in single cells upon stimulation of several GPCRs, including the LPA 2, M 3 and BK 2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gα i1, Gα i2 and Gα i3 activation will be valuable for live-cell measurements that probe Gα i activation.