This report highlights some of the recent developments that have been made in the quantitative analysis of fluorescence decays acquired with pyrene-labeled macromolecules. With these new analytical tools, macromolecules of different composition and architecture can now be labeled in a variety of ways with the pyrene chromophore, and the kinetics of pyrene excimer formation can be described to retrieve quantitative information about the internal dynamics of the macromolecules studied. In particular, this review presents the procedure that was followed to develop these new analytical tools and how the process of pyrene excimer formation with vinyl polymers, poly(L-glutamic acid), dendrimers, associative polymers, surfactants, and lipids labeled with pyrene has been successfully characterized thanks to these analysis programs.