Studies of proteins' interaction in cells by FRET can take benefit from two important photo-physical properties describing fluorescent proteins: fluorescence emission spectrum and fluorescence lifetime. These properties provide specific and complementary information about the tagged proteins and their environment. However, none of them taken individually can completely quantify the involved fluorophore characteristics due to their multiparametric dependency with molecular environment, experimental conditions, and interpretation complexity. A solution to get a better understanding of the biological process implied at the cellular level is to combine the spectral and temporal fluorescence data acquired simultaneously at every cell region under investigation. We present the SLiM-SPRC160, an original temporal/spectral acquisition system for simultaneous lifetime measurements in 16 spectral channels directly attached to the descanned port of a confocal microscope with two-photon excitation. It features improved light throughput, enabling low-level excitation and minimum invasivity in living cells studies. To guarantee a fairly good level of accuracy and reproducibility in the measurements of fluorescence lifetime and spectra on living cells, we propose a rigorous protocol for running experiments with this new equipment that preserves cell viability. The usefulness of SLiM approach for the precise determination of overlapping fluorophores is illustrated with the study of known solutions of rhodamine. Then, we describe reliable FRETexperiments in imaging mode realized in living cells using this protocol. We also demonstrate the benefit of localized fluorescence spectrum-lifetime acquisitions for the dynamic study of fluorescent proteins.