Recently, membrane rafts and caveolae have received much attention for their role as signaling platforms, particularly due to their involvement in the pathogenesis of a number of diseases, including HIV as well as neurological and cardiovascular conditions. Signaling mediated by the beta-adrenergic receptor (beta-AR), a member of the large family of G-protein coupled receptors (GPCRs) that transduce extracellular messages via the ubiquitous second messenger, cAMP, has been a focus of raft studies since multiple components of the pathway are compartmentalized by these membrane microdomains. However, how these membrane rafts behave and regulate signaling dynamics in a cellular context is poorly understood. Here, we describe a live-cell assay based on single-cell, real-time fluorescence imaging, via an improved FRET-based cAMP biosensor, to monitor raft regulation of second messenger dynamics. Upon cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD), beta(2)-AR-mediated cAMP accumulation was enhanced and prolonged in HEK-293 cells, demonstrating that membrane raft integrity helps shape beta-AR signaling. Single-cell imaging in parallel with fractionation studies reveal that the enhancement and change of dynamics are mediated by the receptor and correlated with its redistribution. Finally, the effect of cholesterol depletion is receptor-type specific as MbetaCD treatment did not show the same effect when the raft-excluded prostaglandin E receptor was stimulated. This study highlights the potential of a live-cell, real-time imaging assay for studying membrane rafts, including high sensitivity and spatiotemporal resolution, to achieve a better understanding of the nuances of membrane microdomains in both healthy and diseased states.