We describe a new live-cell assay that uses fluorescent biosensors co-expressed with several GPCRs for measuring the second messenger concentration changes and receptor internalization that occurs after GPCR activation. The molecular structure of the Biosensors comprises: a membrane localization peptide, a second messenger transduction protein binding peptide, a reticulum retention signal and a fluorescent peptide. The biosensors are normally localized in the Plasma Membrane but an increase in the second messenger concentration leads to a change in the structural folding of the Biosensor that promotes its vesicularization. The second messenger transduction protein binding peptide could be replaced depending on the second messenger, resulting three different versions of the biosensors: cAMP; Calcium and DAG Nomad Biosensor. In addition, we also present here the development of multiplexed assays using the different versions of the Biosensor enabling the analysis both different intracellular signaling and receptor internalization, simultaneously.