This thesis presents the development and application of fluorescence lifetime spectroscopy and imaging to the readout of cellular processes using Förster resonant energy transfer (FRET). For quantitative solution-based studies, a multidimensional fluorometer was refined and applied to characterise two genetically encoded calcium FRET biosensors based on Troponin-C, including a fluorescence lifetime-resolved titration study leading to a quantitative calibration of their calcium response. A study of their time-resolved fluorescence anisotropy was also undertaken to explore the potential to probe molecular conformational changes. For the study of signalling processes in live cells, a novel optically sectioning FLIM microscope was developed to provide multiplexed fluorescence lifetime readouts of different FRET probes in order to facilitate the observation of different components of biological signalling networks by realising FLIM interleaved in different spectral channels. This PhD project was motivated to study the AMP-activated protein kinase (AMPK) cascade, which has a central role in the regulation of the energy level in mammalian cells and is now being studied as a potential drug target for type II diabetes. One pathway leading to activation of AMPK is triggered by an increase in intracellular calcium level leading to activation of calcium-calmodulin dependent protein kinase kinase β (CaMKKβ), an upstream activator of AMPK. This was investigated through a novel inter-molecular FRET system looking at the direct interaction of AMPK and CaMKKβ. The ultimate goal was to multiplex readouts of AMPK activation and intracellular calcium levels, for which new FRET biosensors are required. To this end, work was also undertaken for the design and production of novel FRET sensors in the red part of the spectrum, which are desirable for multiplexing with the common CFP/YFP–based FRET probes and also for use in in vivo imaging applications.