Abstract: A quantitative fluorescence technique is presented as "differential real-time fluorescence spectroscopy" (DRFS), pursuant to which one can simultaneously make time-resolved measurement of fluorescence changes in two cuvette samples (e.g., experimental and control), wherein the fluorescence is induced by excitation light, and wherein both samples have been identically prepared with a selected ratiometric dye. The ratiometric dye may either be of excitation-shifted variety or of emission-shifted variety. With DRFS, it is possible to monitor the response of specimen cells to a chemical, biological or physical agent, in the absence and in the presence of a selected experimental variable, and to determine both of these responses in real time, i.e., at the same time as the measurements are being made.