Background: Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in F⏠orster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed. Methods: We use known fluorophores to perform characterization experiments in our instrumental setup. This allows us to verify the accuracy of the fluorescence lifetime determination, and test the linearity of the instrument by fluorescence quenching. Results: We develop and validate here a protocol for rigorous characterization of time-domain FLIM instruments. Following this protocol, we show that our system pro-