An in vitro system for 2.45 GHz microwave (MW) exposure with real-time fluorescence measurements is proposed. This system is specifically designed for the measurement of those biophysical parameters of living cells or membrane models which can be quantified by spectrofluorometric methods (e.g. membrane generalized polarization (GP), membrane fluidity, membrane potential, etc.). The novelty of the system consists in the possibility to perform fluorescence measurements on the biological samples simultaneously with their exposure to MW. The MW applicator is an open ended coaxial antenna which is dipped into a cuvette. The distribution of electromagnetic field and specific absorption rate (SAR) in the cuvette are provided from a rigorous electromagnetic numerical analysis performed with a finite difference-time domain (FDTD) based tool. With this system, fluorescence measurements were used to calculate the membrane GP values of giant unilamellar vesicle suspensions that were acquired during exposure to a 1.2 W incident power. For this power, the SAR distribution and mean SAR value for the whole volume were calculated based on temperature measurements made at different positions inside the cuvette.