Fluorescein is a complex fluorophore which at the physiological pH can exist as mono- and dianion forms. In a previous article (J. Phys. Chem. A 2001, 105, 6320-6332), we showed that fluorescein displays an excited-state proton transfer reaction which interconverts the mono-and dianion forms in the presence of a suitable proton donor-acceptor. Because fluorescein is a frequently used fluorescent label in biological systems, it is of interest to know if amino acids with acidic side chains are able to induce fluorescein excited-state proton transfer reactions. We have selected (+/-)-N-acetyl aspartic acid, N-AcAsp, as a model donor-acceptor which mimics the interaction of the aspartic acid residues with the fluorescent label in native proteins. We present absorption and emission properties which show that N-AcAsp at I M concentration induces the excited-state proton transfer reaction between the mono- and dianion of fluorescein. The kinetics of this excited-state process was characterized from time-resolved fluorescence measurements, and the analysis was done within the framework of global compartmental analysis. Thus, we have formulated the fluorescence decay analysis of intermolecular two-state excited-state processes in the presence of buffer in terms of compartments. The kinetic equations describing the excited-state species concentrations were derived, and the expressions for the fluorescence decay surface were obtained. The experimental fluorescence decay data surface of fluorescein in the presence of 1 M N-AcAsp concentration at different pH values and excitation and emission wavelengths was analyzed via this new global compartmental analysis method, to recover the rate constants and the spectral parameters related to absorption and emission of the excited-state proton transfer reaction.