G-protein-coupled receptors transmit signals through cascades of cellular signaling molecules to orchestrate physiological responses. Measuring the time course (kinetics or dynamics) of GPCR signaling is revealing new spatiotemporal paradigms of signaling with biological and therapeutic implications. Kinetics also impact drug activity measurements, for example biased agonism. Biosensors greatly facilitate the measurement of signaling kinetics by detecting signaling in live cells in real time; a ligand is applied to cells, the cells placed in a reader, and the change in light emission measured repeatedly over time. In this chapter, methods are presented for assaying signaling molecules using genetically-encoded fluorescent biosensors. Protocols are provided for the most common G-protein pathways (Gs and Gi, detecting cAMP; Gq, detecting diacylglycerol and Ca2+), and also for arrestin recruitment. Methods for analyzing the time course data are required to obtain kinetic drug signaling parameters useful for lead optimization and large-scale receptor research. One such parameter is the initial rate of signaling by the ligand bound receptor (kτ), a biologically-meaningful and familiar metric. Straightforward curve fitting methods are described for measuring the initial rate by analyzing the whole time course without the need to isolate the linear portion of the curve. Biased agonism is quantified using this approach, providing a simple metric that takes into account differential kinetics of signaling. These experimental and analysis methods enable investigators to routinely measure and quantify the kinetics of signaling in their receptor research and drug discovery projects.